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Gossypol-induced DNA breaks in rat lymphocytes are secondary to cytotoxicity
Toxicology Letters 117 (2000) 85 – 94 www.elsevier.com/locate/toxlet
Gossypol-induced DNA breaks in rat lymphocytes are secondary to cytotoxicity Penelope J.E. Quintana *, Ann de Peyster, Stephen Klatzke, Hyun Jung Park Di6ision of Occupational and En6ironmental Health, Graduate School of Public Health, San Diego State Uni6ersity, 5500 Campanile Dr. HT-119, San Diego, CA 92182 -4162, USA Received 20 March 2000; received in revised form 21 July 2000; accepted 21 July 2000
Abstract Gossypol, a male antifertility and potential anticancer agent, was found to induce DNA strand breaks in rat lymphocytes. DNA breaks were measured with the single-cell gel electrophoresis (SCGE) or ‘comet’ assay. A significant increase in DNA breaks was observed after 1 h incubation at concentrations of 2 mg/ml or greater. The inclusion of 10% fetal bovine serum in the media reduced the toxicity of gossypol, and DNA breaks were only observed at a concentration of 80 mg/ml. However, the increase in DNA strand breaks, for incubations with and without serum, only occurred when cell viability was reduced to less than 70%. Examination of cell morphology and DNA fragmentation at incubations up to 5 h yielded no evidence that DNA strand breaks were occurring due to apoptosis. We conclude that gossypol is not primarily genotoxic in this cell type, and that the DNA breaks observed arose secondary to cytotoxicity. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gossypol; DNA breaks; Genotoxicity; SCGE assay; Comet assay; Lymphocytes; Rat
1. Introduction Gossypol is a polyphenolic binaphthalene compound isolated from cotton plants (Gossypium sp., Malvacae) which has several proposed clinical applications. Many investigators have explored its potential for use as an oral, nonsteroidal male contraceptive agent following the discovery by
* Corresponding author. Tel.: +1-619-5941688; fax: +1619-5946112. E-mail address: [email protected] (P.J.E. Quintana).
Chinese scientists that reversible inhibition of spermatogenesis is one of the earliest effects observed at low doses in humans (Segal, 1985), although its use appears to be limited by a 10% chance of permanent aspermia and occasional cases of hypokalemia (Waites et al., 1998). More recently, limited clinical trials suggest that gossypol also holds promise as a treatment for adrenal, prostate, and mammary carcinomas, gliomas, endometriosis and uterine myoma (Han et al., 1987; Stein et al., 1992; Flack et al., 1993; Bushunow et al., 1999). Lack of myelotoxicity and other typical serious side effects of chemotherapy were also
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considered encouraging findings in one clinical trial (Stein et al., 1992). Further characterization of the safety and potential side effects of gossypol and understanding its mechanism(s) of action are important for possible pharmaceutical development. A review of genetic toxicity studies on gossypol had suggested a weak genotoxic potential under normal physiologic conditions (de Peyster and Wang, 1993). Gossypol was reported to induce DNA strand breaks in both human fibroblasts and human leukocytes treated in vitro (Nordenskjold and Lambert, 1984; Chen et al., 1986). Also, gossypol exposure resulted in weak positive or equivocal results in tests of sister chromatid exchange (SCE) frequency (Nordenskjold and Lambert, 1984; Nayak and Buttar, 1986; Wang et al., 1988). However, gossypol was not mutagenic in the Ames Salmonella test (de Peyster and Wang, 1979; Majumdar et al., 1982) and did not induce micronuclei or chromosome aberrations in vivo or in vitro in mammalian systems (de Peyster and Wang, 1993). The overall objective of the present experiments was to gain a better understanding of the genotoxic effects previously reported for gossypol, using a sensitive assay for DNA strand breaks, the single cell gel electrophoresis (SCGE) or ‘comet’ assay (Singh et al., 1988, 1994). In previous studies of DNA strand breaks using alkaline elution or hydroxlyapatite (Nordenskjold and Lambert, 1984; Chen et al., 1986), the DNA breaks appeared only at gossypol concentrations that potentially approached cytotoxic levels. In order to assess whether the DNA breaks reported after exposure to gossypol might actually have been secondary to cytotoxicity, rather than true genotoxic damage occurring in viable cells, we measured DNA strand breaks and cytotoxicity in rat lymphocytes following a 1 h in vitro exposure to gossypol. We also investigated whether the DNA strand breaks reported might be due to apoptosis by assessing morphological characteristics and the pattern of DNA damage in the SCGE assay after 1, 3, and 5 h incubations with gossypol.
2. Materials and methods
2.1. Materials Gossypol acetic acid (CAS 12542-36-8) was purchased from Sigma (St. Louis, MO), as were all other chemicals and reagents used in these experiments, with the following exceptions, serum free RPMI Media 1640 with L-glutamine and 2.0 g/l sodium bicarbonate (media) was purchased from the Core Cell Culture Facility at the University of California at San Diego (San Diego, CA), and D-erythrosphingosine was obtained from Calbiochem-Novabiochem Corporation (San Diego, CA). Gossypol acetic acid is referred to in this paper as gossypol, but our concentrations and molarities are reported for gossypol acetic acid (1.0 mg/ml =1.73 mM).
2.2. Rat lymphocyte isolation Tail vein or trunk blood from anesthetized Sprague–Dawley male rats (approximately 350– 450 g) was collected into heparinized Vacutainer™ tubes. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee. Blood samples were layered onto HISTOPAQUE™ and centrifuged for 30 min at 400× g at room temperature. The opaque interface was collected, washed 2× , and resuspended in RPMI media with 10% heat-inactivated fetal bovine serum (FBS). Lymphocyte viability was assessed by scoring at least 100 cells for Trypan Blue exclusion (0.4% at 1:5 dilution).
2.3. Treatment of cells Cells were suspended at a concentration of 5× 105 cells per ml for treatments. Gossypol solutions were prepared in dimethylsulfoxide (DMSO) immediately prior to use. Gossypol concentrations ranging from 0.01 to 10 mg/ml were tested in the absence of FBS, and concentrations from 10 to 80 mg/ml were tested in the presence of 10% FBS. DMSO was used as the vehicle control, and the final concentration was always less than 2%. Cells were incubated at 37°C and 5% CO2, and
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viability was assessed by Trypan Blue exclusion for each time point at which SCGE slides were prepared. Positive controls were sphingosine (5 mM) for apoptosis (Sweeney et al., 1996) and H2O2 (30 mM) for DNA single-strand breaks (Fairbairn et al., 1995).
2.4. Measurement of DNA damage by SCGE assays The alkaline SCGE assay was performed essentially as described in Singh et al. (1994) and all steps were conducted under dimmed light (Anderson et al., 1994). Briefly, after each incubation, 10 000 cells from each treatment group were mixed with 75 ml of 0.5% low melting point agarose in phosphate buffered saline (PBS), and spread on a microscope slide coated with 1.0% normal melting point agarose in Ca2 + and Mg2 + free PBS. A top layer of 0.5% low melting point agarose in PBS was added and the slides immersed in lysis buffer solution (2.5 M NaOH, 100 mM ethylene diamine tetraacetic acid (EDTA), 10 mM Tris Base, 1% N-laurylsarcosine, 1% Triton X-100 and 10% DMSO, pH 10) for 1 h at 4°C. Slides were then placed in electrophoresis buffer (1 mM EDTA, 300 mM NaOH, and 0.1% 8-hydroxyquinoline), left for 20 min, then electrophoresed for 20 min at 2 V/cm. Slides were neutralized with 0.4 M Tris Base, pH 7.5 and preserved by drying in 95% ethanol. DNA migration was visualized by staining with 10 mg/ml ethidium bromide and examining 50 nuclei per slide using an Olympus BH2-RFL epifluorescent microscope and ethidium bromide filter cube (Olympus America Inc., Melville, NY) connected to a Pulnix TM-745E CCD camera (Pulnix Inc., Sunnyvale, CA). A computerized image analysis system, Komet, version 3.1 (Kinetic Imaging Ltd., Liverpool, UK), and Windows, version 3.1 (Microsoft Corporation, Seattle, WA) were used to measure nuclear parameters. Parameters used were tail extent moment (percent DNA in the tail × tail length) and Olive tail moment (percent DNA in the tail × mean tail displacement). Three or more independent experiments were performed for each exposure condition and duplicate slides were made for each exposure. Non-parametric
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Kruskal–Wallis and Mann–Whitney U-tests were used to test for statistical significance among treatments, using the SPSS statistical package (SPSS Inc., Chicago, IL). A P-value of 5 0.05 was considered significant.
2.5. Measurement of cell morphology by hoescht/propidium iodide staining and microscopy Morphological features consistent with apoptosis were assessed through the following modification of the procedure used in Sun et al. (1992), Krown et al. (1996). Following incubations, cells were stained with 0.1 mg/ml hoescht bisbenzimide c 33342 for 5 min, then with 0.5 mg/ml propidium iodide (PI). Cells were scored under fluorescence microscopy (Olympus BX40 System microscope with a BX-FLA fluorescence attachment, DAPI and PI filter cube set) for condensed, pyknotic nuclei and membrane features consistent with apoptosis (Majno and Joris, 1995). Membrane integrity was assessed by PI exclusion, 20–50 cells were scored per treatment. Two or more independent experiments were performed for each treatment and duplicate cultures were made for each exposure.
3. Results
3.1. Cytotoxicity of gossypol after 1 h incubation with and without serum in media Lymphocytes were exposed from 0.01 to 10 mg/ml in media without FBS and from 10 to 80 mg/ml in media with FBS. When lymphocytes were exposed to gossypol without serum in the media, gossypol concentrations of 1 mg/ml or higher were found to cause cytotoxic effects and exposures of 10 mg/ml resulted in 100% loss of viability (Fig. 1). We found that gossypol-induced cytotoxicity was reduced in the presence of serum. For most experiments with serum added, gossypol concentrations of 10–40 mg/ml produced measured viability similar to controls, with ctyotoxic effects seen at 60 mg/ml and above. However, the variability in response to gossypol was greater in media with serum than for serum-free media, and
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in some of these experiments the viability was much reduced.
3.2. DNA strand breaks after 1 h incubation with gossypol with and without serum in media
Viability as percent of control
To assess whether gossypol induced DNA strand breaks in the absence of cytotoxicity, we performed the SCGE assay on cells after 1 h incubations with gossypol. Fig. 2A depicts nuclei from peripheral lymphocytes treated with DMSO only, which exhibited very little DNA fragmentation after electrophoresis. Fig. 2B, in contrast, shows DNA damage in the form of a ‘comet tail’ extending away from the nucleus in cells exposed to 2 mg/ml gossypol. The amount of DNA strand breakage was recorded as the ‘tail extent moment’ (TM), which is the product of the length of the tail and the percent of total DNA from that nucleus that is in the tail region. Table 1A shows the average TM from cells incubated with 0.5, 1.0,
and 2.0 mg/ml gossypol in serum-free media for 1 h. Gossypol was found to increase DNA strand breaks by 7–8 fold over control at concentrations of 2.0 mg/ml, but at this dose cell viability was reduced to less than 50%. Concentrations higher than 2.0 mg/ml caused 100% damaged, unscorable nuclei, and were not tested in the SCGE assay after initial experiments (data not shown). In contrast, H2O2 produced extensive DNA breaks even though cell viability remained high ( \ 95%, Table 1A). We were unable to detect DNA damage caused by gossypol under conditions where cell viability was similar to controls. The production of DNA strand breaks by gossypol was reduced or not seen in the presence of serum proteins. After incubation with gossypol concentrations of 20–80 mg/ml with fetal bovine serum in the media, gossypol induced significant DNA damage only at 80 mg/ml (Table 1B). At this concentration the viability was also affected, being an average of 23%. However, in a few
100%
80%
60%
(-) Series1
(i+) Ser es2
FBS
FBS
40%
20%
0% 0.01
0.1
1
10
100
pol ac C oncentration of go ssy etic aci d ( g/m l) Fig. 1. Cytotoxicity of gossypol at 1 h incubation. Rat lymphocytes were incubated with gossypol at indicated concentrations, both with (left line) and without (right line) FBS in media. Cell viability was assessed by Trypan Blue exclusion (Section 2.1) and is shown as percentage of control (cells incubated with DMSO vehicle control only). Error bars depict standard error of the mean. (FBS, fetal bovine serum, DMSO, dimethylsulfoxide).
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A.
B.
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3.3. DNA strand breaks and cellular morphology after 5 h incubation with gossypol To investigate whether some of the strand breakage observed in earlier studies could be secondary to apoptosis, incubations were carried out for longer times. Serum was included in the media, to preclude apoptosis caused by serum withdrawal. DNA damage was assessed in the SCGE assay and recorded as the Olive tail moment (OliveTM), which is a measure useful for discriminating apoptosis and accidental cell death (Fairbairn et al., 1996). Fig. 3 shows image analysis data of comets from cells exposed to gossypol at Table 1 Induction of cytotoxicity and DNA strand breaks by goosypol in rat lymphocytes (1 h incubation) Treatment Cell viability serum-free (1 h) (mean 9S.D., %)
DNA breaks (TM, mean 9S.D.)c
(a) Treatments in serum free media
Fig. 2. Images of ethidium bromide-stained nuclei from rat lymphocyte cells processed in the comet assay. (A) Undamaged nuclei from cells exposed to DMSO, the vehicle control. (B) Nuclei from cells exposed to gossypol at 2 ug/ml for 1 h as described in Section 2.3. The ‘comet tail’ extends to the positive electrode during electrophoresis. These nuclei exhibit moderate to severe DNA strand breakage; which was quantified by image analysis as described in Section 2.4.
experiments where cell viability was much reduced (two out of nine experiments), we did observe significant DNA damage at lower concentrations of gossypol. For example, in one such experiment, the viability was much lower than average (55, 25, and 18% at concentrations of 20, 40 and 60 mg/ml respectively), and statistically significant increases in TM were seen in the 40 and 60 mg/ml gossypol treatment groups. This confirms that although gossypol can induce DNA damage, the DNA damage was never significantly greater than controls except when accompanied by a reduction in cell viability.
Control DMSO onlyd
99 92
Gossypol 0.5 mg/ml 0.1 mg/ml 2.0 mg/ml
97 90.5 90 9 2 51 9 10b
3 9 17 49 2.4 396.0 239 12.6b
Hydrogen peroxide (H2O2) 30 mM 959 2
165 9 85b
Treatment with serum (1 h)
DNA breaks (TM, mean 9S.D.)c
Cell viability (mean 9S.D., %)
(b) Treatments in media with 10% fetal bo6ine serum Control 98 9 1 1.5 92.8 DMSO onlyd Gossypol 10 mg/ml 40 mg/ml 80 mg/ml
95 9 2 83 925 23 9 39
Hydrogen peroxide (H2O2) 30 mM 89 9 5 a
0.62 91.7 11 922 33933a 839 25b
PB0.5. PB0.0001 (Kruskal–Wallis test followed by Mann–Whitney U-test). c TM, tail extent moment = (comet tail length)×(comet tail percent DNA/100). d DMSO, dimethylsulfoxide. b
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mainly consequent to accidental cell death associated with membrane permeability.
4. Discussion
Fig. 3. DNA fragmentation after a 5 h incubation with gossypol. Rat lymphocytes were incubated with gossypol for 5 h at indicated concentrations in media containing FBS. DNA damage as assessed by the comet assay is plotted with cell viability as measured by Trypan Blue exclusion. DNA damage is recorded as the Olive tail moment (OliveTM) as described in Section 2.4.
20, 40 and 60 mg/ml for 5 h, along with cell viability data. Both the 40 and 60 mg/ml concentrations of gossypol produced a significant increase in OliveTM. However, these concentrations were associated with a significant loss of cell viability, as assessed by Trypan Blue exclusion. The sphingosine-treated cells exhibit a significant increase in DNA damage expressed as a high OliveTM, but little loss of cell membrane permeability (viability \ 70%); a pattern associated with early apoptosis (Majno and Joris, 1995). Assessment of cell and nuclear morphological changes were carried out to confirm that gossypol-induced DNb A strand breaks were not being produced as a result of apoptotic mechanisms. Lymphocytes were exposed 10 – 80 mg/ml gossypol and scored at 1, 3 and 5 h for membrane changes and condensed, pyknotic nuclei associated with apoptosis, using a combination Hoescht/propidium iodide stain. No effect on morphology was seen, although membrane viability was affected as early as 1 h at the high dose (Fig. 4). We therefore, conclude that DNA strand breaks are
Our main finding was that genetic toxicity of gossypol in the SCGE assay is not significant in the absence of cytotoxicity. When cell viability after gossypol treatment was higher than 70% we did not detect a significant increase in DNA strand breaks, either with or without serum in the media. The concentrations of gossypol that induced DNA strand breaks in the presence of cytotoxicity were 2 mg/ml (3.5 mM) and greater without serum in the media and 80 mg/ml and greater when 10% FBS was present in the media (Table 1). The relationship between cytotoxicity and genotoxicity has been the focus of some discussion. In the SCGE assay, necrosis resulting from accidental cell death has been shown to produce fragmented DNA that can mimic genotoxicity or
Fig. 4. Nuclear morphology and viability of rat lymphocytes assessed after 1 and 5 h incubations with gossypol. Rat lymphocytes were incubated with gossypol for 5 h at indicated concentrations in media containing FBS. Condensed, pyknotic nuclei and cell viability were scored as describect in Section 2.5.
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apoptotic damage (Fairbairn and O’Neill, 1995). This observation has led Henderson et al. (1998) suggest that a cut off of 75% viability be used in the SCGE assay to discriminate between ‘true’ genotoxicity and genotoxicity secondary to cytotoxicity. By this criterion, gossypol would not be considered genotoxic in rat lymphocytes as assessed by the work reported here using the SCGE assay. The conclusions from our study are in contrast to the conclusions reached in some previous studies which reported that gossypol induced DNA strand breaks in human fibroblasts and human leukocytes, and concluded that gossypol is genotoxic. The other studies used methods thought to be less sensitive than the SCGE assay (Leroy et al., 1996). Chen et al. (1986) found a dose-dependent increase of DNA single strand breaks as measured by alkaline elution and hydroxylapatite chromatography, when human leukocytes were exposed to 1–15 mg/ml of gossypol for 1 h in serum-free medium. These authors also reported that DNA strand breaks were decreased in cells treated with gossypol in the presence of 10% FCS compared with serum-free media. Nordenskjold and Lambert (1984) had observed DNA single strand breaks using the method of hydroxylapatite chromatography, when human skin fibroblasts were exposed to gossypol at concentrations of 5 –40 mg/ml in serum-free PBS for 30 min at 37°C. Cell viability was not reported. The effect was not seen for a 20 mg/ml exposure in the presence of 2% fetal calf serum. They also measured DNA repair by removing the gossypol after 30 min and following the single strand breaks over time. No repair was seen over 5 h, which is consistent with the DNA breaks being secondary to cytotoxicity. We also explored the possibility that strand breaks might be due to apoptosis by examining DNA damage at further time periods beyond 1 h, as rat spermatocytes showed DNA fragmentation and laddering when exposed to gossypol for 5 h at a concentration of 100 mM (58 mg/ml) and higher (Teng, 1995). Jarvis et al. (1994) also has reported apoptosis-like DNA fragmentation at concentrations of 50–100 mM in HL-60 cells when incubated for 6 h. Shelley et al. (1999) reported DNA
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fragmentation and cell changes consistent with apoptosis in a melanoma cell line exposed to the l-enantiomer of gossypol. We did not find any evidence of apoptosis as assessed by the SCGE assay and cell morphology after a 5 h incubation, and we clearly observed DNA damage from gossypol concomitant with reduced cell viability after a 1 h exposure, which is unlikely to have been due to apoptotic scission (Figs. 3 and 4). It may be that lymphocytes are not susceptible to gossypol-induced apoptosis, unlike other cell types tested, such as cancer cell lines. Teng (1998) also reported reduced viability (67% and lower) at concentrations producing features of apoptosis, so some of the DNA breakage may not have been due to apoptosis but to necrosis. We used lymphocytes as the cell type tested in this study in order to have a primary, untransformed cell type comparable with the cells used in the studies reporting positive results for gossypol-induced DNA strand breaks. A number of theories are postulated to explain the toxic and therapeutic effects of gossypol. Gossypol reacts with many cell macromolecules, binding covalently to epsilon-amino acids through Schiff’s base condensation reactions (Adams et al., 1960). Many of the disruptive effects seen on enzymes and other proteins could be explained by this general mechanism (Strom-Hansen et al., 1989). The many enzymes already known to be inhibited by gossypol (adenylate cyclase, acrosin, protein kinase C, LDH, glutathione-S-transferase, to name a few) have been reviewed by Qian and Wang (1984). Gossypol also chelates iron, copper, aluminum and zinc (Berardi and Goldblatt, 1969); inhibits mitochondrial oxidative phosphorylation (Abou-Donia and Dieckert, 1974); manifests both pro-oxidant and antioxidant characteristics (Coburn et al., 1980; de Peyster et al., 1984; Sheriff et al., 1986; Bender et al., 1988; Janero and Burghardt, 1988; Grankvist, 1989; Laughton et al., 1989), alters membrane potential, fluidity and permeability (Reyes et al., 1984; de Peyster et al., 1986; Benz et al., 1991). and disrupts cell-tocell communication at doses below those necessary to cause appreciable cytotoxicity (Ye et al., 1990; Herve et al., 1996). The vast majority of in vitro studies also report that the presence of
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serum proteins reduces or eliminates the effect of gossypol (see for example Haspel et al., 1984; Hu et al., 1993). Some of the earliest ultrastructural damage occurs in the mitochondrial membrane (Hu et al., 1986; Benz et al., 1990). A study detailing the timing of gossypol-induced toxicity implicates the generation of reactive oxygen species as the primary event (Barhoumi and Burghardt, 1996). Tuszynski and Cossu (1984) and Band et al. (1989) measured cell viability and 3 H-thymidine uptake in cells and observed that effects on DNA synthesis appear after a significant increase in membrane permeability has occurred. This type of evidence suggests that the primary effect of gossypol is interference with one or more non-nuclear cell functions. In summary, a statistically significant increase in DNA strand breaks after exposure to gossypol was demonstrated here. However, DNA strand breaks only occurred in the presence of cytotoxicity, where cell viability was affected. We therefore conclude that gossypol-induced genotoxicity is likely secondary to cytotoxicity in this cell type. This information helps to characterize the safety of gossypol for various clinical uses, as well as to contribute to understanding mechanisms of its toxicity to tumor and non-target cells.
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Nordenskjold, M., Lambert, B., 1984. Gossypol induces DNA strand breaks in human fibroblasts and sister chromatid exchanges in human lymphocytes in vitro. J. Med. Genet. 21, 129 – 132. Qian, S.Z., Wang, Z.G., 1984. Gossypol: a potential antifertility agent for males. Annu. Rev. Pharmacol. Toxicol. 24, 329 – 360. Reyes, J., Allen, J., Tanphaichitr, N., Bellve, A.R., Benos, D.J., 1984. Molecular mechanisms of gossypol action on lipid membranes. J. Biol. Chem. 259, 9607 – 9615. Segal, S.J., 1985. Gossypol, a Potential Contraceptive for Men. Plenum Press, New York. Shelley, M.D., Hartley, L., Fish, R.G., Groundwater, P., Morgan, J.J., Mort, D., Mason, M., Evans, A., 1999. Stereo-specific cytotoxic effects of gossypol enantiomers and gossypolone in tumour cell lines. Cancer Lett. 135, 171 – 180. Sheriff, D.S., el Fakhri, M., Ghwarsha, K., Elgengan, K.B., 1986. Gossypol and lipid peroxidation. Am. J. Obstet. Gynecol. 155, 457 – 458. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184 – 191. Singh, N.P., Stephens, R.E., Schneider, E.L., 1994. Modifications of alkaline microgel electrophoresis for sensitive detection of DNA damage. Int. J. Radiat. Biol. 66, 23 – 28. Stein, R.C., Joseph, A.E., Matlin, S.A., Cunningham, D.C., Ford, H.T., Coombes, R.C., 1992. A preliminary clinical study of gossypol in advanced human cancer. Cancer Chemother. Pharmacol. 30, 480 – 482. Strom-Hansen, T., Cornett, C., Jaroszewski, J.W., 1989. Interaction of gossypol with amino acids and peptides as a model of enzyme inhibition. Int. J. Pept. Protein Res. 34, 306 – 310. Sun, X.M., Snowden, R.T., Skilleter, D.N., Dinsdale, D., Ormerod, M.G., Cohen, G.M., 1992. A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes. Anal. Biochem. 204 (2), 351 – 356. Sweeney, E.A., Sakakura, C., Shirahama, T., Masamune, A., Ohta, H., Hakomori, S., Igarashi, Y., 1996. Sphingosine and its methylated derivative N,N-dimethylsphingosine (DMS) induce apoptosis in a variety of human cancer cell lines. Int. J. Cancer 66, 358 – 366. Teng, C.S., 1995. Gossypol-induced apoptotic DNA fragmentation correlates with inhibited protein kinase C activity in spermatocytes. Contraception 52, 389 – 395. Teng, C.S., 1998. c-fos Protein expression in apoptotic rat spermatocytes induced by gossypol. Contraception 57, 281 – 286. Tuszynski, G.P., Cossu, G., 1984. Differential cytotoxic effect of gossypol on human melanoma, colon carcinoma, and other tissue culture cell lines. Cancer Res. 44, 768 – 771. Waites, G.M., Wang, C., Griffin, P.D., 1998. Gossypol: reasons for its failure to be accepted as a safe, reversible male antifertility drug. Int. J. Androl. 21, 8 – 12. Wang, R.L., Tan, Y.B., Zhang, Z.S., 1988. Gossypol acetate-
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induced SCEs in spermatogonia and bone marrow cells of mice: dose-response relationships. Contraception 37, 191– 195. Ye, Y.X., Bombick, D., Hirst, K., Zhang, G.X., Chang, C.C.,
Trosko, J.E., Akera, T., 1990. The modulation of gap junctional communication by gossypol in various mammalian cell lines in vitro. Fundam. Appl. Toxicol. 14, 817 – 832.
.
Gossypol-induced DNA breaks in rat lymphocytes are secondary to cytotoxicity Penelope J.E. Quintana *, Ann de Peyster, Stephen Klatzke, Hyun Jung Park Di6ision of Occupational and En6ironmental Health, Graduate School of Public Health, San Diego State Uni6ersity, 5500 Campanile Dr. HT-119, San Diego, CA 92182 -4162, USA Received 20 March 2000; received in revised form 21 July 2000; accepted 21 July 2000
Abstract Gossypol, a male antifertility and potential anticancer agent, was found to induce DNA strand breaks in rat lymphocytes. DNA breaks were measured with the single-cell gel electrophoresis (SCGE) or ‘comet’ assay. A significant increase in DNA breaks was observed after 1 h incubation at concentrations of 2 mg/ml or greater. The inclusion of 10% fetal bovine serum in the media reduced the toxicity of gossypol, and DNA breaks were only observed at a concentration of 80 mg/ml. However, the increase in DNA strand breaks, for incubations with and without serum, only occurred when cell viability was reduced to less than 70%. Examination of cell morphology and DNA fragmentation at incubations up to 5 h yielded no evidence that DNA strand breaks were occurring due to apoptosis. We conclude that gossypol is not primarily genotoxic in this cell type, and that the DNA breaks observed arose secondary to cytotoxicity. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gossypol; DNA breaks; Genotoxicity; SCGE assay; Comet assay; Lymphocytes; Rat
1. Introduction Gossypol is a polyphenolic binaphthalene compound isolated from cotton plants (Gossypium sp., Malvacae) which has several proposed clinical applications. Many investigators have explored its potential for use as an oral, nonsteroidal male contraceptive agent following the discovery by
* Corresponding author. Tel.: +1-619-5941688; fax: +1619-5946112. E-mail address: [email protected] (P.J.E. Quintana).
Chinese scientists that reversible inhibition of spermatogenesis is one of the earliest effects observed at low doses in humans (Segal, 1985), although its use appears to be limited by a 10% chance of permanent aspermia and occasional cases of hypokalemia (Waites et al., 1998). More recently, limited clinical trials suggest that gossypol also holds promise as a treatment for adrenal, prostate, and mammary carcinomas, gliomas, endometriosis and uterine myoma (Han et al., 1987; Stein et al., 1992; Flack et al., 1993; Bushunow et al., 1999). Lack of myelotoxicity and other typical serious side effects of chemotherapy were also
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considered encouraging findings in one clinical trial (Stein et al., 1992). Further characterization of the safety and potential side effects of gossypol and understanding its mechanism(s) of action are important for possible pharmaceutical development. A review of genetic toxicity studies on gossypol had suggested a weak genotoxic potential under normal physiologic conditions (de Peyster and Wang, 1993). Gossypol was reported to induce DNA strand breaks in both human fibroblasts and human leukocytes treated in vitro (Nordenskjold and Lambert, 1984; Chen et al., 1986). Also, gossypol exposure resulted in weak positive or equivocal results in tests of sister chromatid exchange (SCE) frequency (Nordenskjold and Lambert, 1984; Nayak and Buttar, 1986; Wang et al., 1988). However, gossypol was not mutagenic in the Ames Salmonella test (de Peyster and Wang, 1979; Majumdar et al., 1982) and did not induce micronuclei or chromosome aberrations in vivo or in vitro in mammalian systems (de Peyster and Wang, 1993). The overall objective of the present experiments was to gain a better understanding of the genotoxic effects previously reported for gossypol, using a sensitive assay for DNA strand breaks, the single cell gel electrophoresis (SCGE) or ‘comet’ assay (Singh et al., 1988, 1994). In previous studies of DNA strand breaks using alkaline elution or hydroxlyapatite (Nordenskjold and Lambert, 1984; Chen et al., 1986), the DNA breaks appeared only at gossypol concentrations that potentially approached cytotoxic levels. In order to assess whether the DNA breaks reported after exposure to gossypol might actually have been secondary to cytotoxicity, rather than true genotoxic damage occurring in viable cells, we measured DNA strand breaks and cytotoxicity in rat lymphocytes following a 1 h in vitro exposure to gossypol. We also investigated whether the DNA strand breaks reported might be due to apoptosis by assessing morphological characteristics and the pattern of DNA damage in the SCGE assay after 1, 3, and 5 h incubations with gossypol.
2. Materials and methods
2.1. Materials Gossypol acetic acid (CAS 12542-36-8) was purchased from Sigma (St. Louis, MO), as were all other chemicals and reagents used in these experiments, with the following exceptions, serum free RPMI Media 1640 with L-glutamine and 2.0 g/l sodium bicarbonate (media) was purchased from the Core Cell Culture Facility at the University of California at San Diego (San Diego, CA), and D-erythrosphingosine was obtained from Calbiochem-Novabiochem Corporation (San Diego, CA). Gossypol acetic acid is referred to in this paper as gossypol, but our concentrations and molarities are reported for gossypol acetic acid (1.0 mg/ml =1.73 mM).
2.2. Rat lymphocyte isolation Tail vein or trunk blood from anesthetized Sprague–Dawley male rats (approximately 350– 450 g) was collected into heparinized Vacutainer™ tubes. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee. Blood samples were layered onto HISTOPAQUE™ and centrifuged for 30 min at 400× g at room temperature. The opaque interface was collected, washed 2× , and resuspended in RPMI media with 10% heat-inactivated fetal bovine serum (FBS). Lymphocyte viability was assessed by scoring at least 100 cells for Trypan Blue exclusion (0.4% at 1:5 dilution).
2.3. Treatment of cells Cells were suspended at a concentration of 5× 105 cells per ml for treatments. Gossypol solutions were prepared in dimethylsulfoxide (DMSO) immediately prior to use. Gossypol concentrations ranging from 0.01 to 10 mg/ml were tested in the absence of FBS, and concentrations from 10 to 80 mg/ml were tested in the presence of 10% FBS. DMSO was used as the vehicle control, and the final concentration was always less than 2%. Cells were incubated at 37°C and 5% CO2, and
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viability was assessed by Trypan Blue exclusion for each time point at which SCGE slides were prepared. Positive controls were sphingosine (5 mM) for apoptosis (Sweeney et al., 1996) and H2O2 (30 mM) for DNA single-strand breaks (Fairbairn et al., 1995).
2.4. Measurement of DNA damage by SCGE assays The alkaline SCGE assay was performed essentially as described in Singh et al. (1994) and all steps were conducted under dimmed light (Anderson et al., 1994). Briefly, after each incubation, 10 000 cells from each treatment group were mixed with 75 ml of 0.5% low melting point agarose in phosphate buffered saline (PBS), and spread on a microscope slide coated with 1.0% normal melting point agarose in Ca2 + and Mg2 + free PBS. A top layer of 0.5% low melting point agarose in PBS was added and the slides immersed in lysis buffer solution (2.5 M NaOH, 100 mM ethylene diamine tetraacetic acid (EDTA), 10 mM Tris Base, 1% N-laurylsarcosine, 1% Triton X-100 and 10% DMSO, pH 10) for 1 h at 4°C. Slides were then placed in electrophoresis buffer (1 mM EDTA, 300 mM NaOH, and 0.1% 8-hydroxyquinoline), left for 20 min, then electrophoresed for 20 min at 2 V/cm. Slides were neutralized with 0.4 M Tris Base, pH 7.5 and preserved by drying in 95% ethanol. DNA migration was visualized by staining with 10 mg/ml ethidium bromide and examining 50 nuclei per slide using an Olympus BH2-RFL epifluorescent microscope and ethidium bromide filter cube (Olympus America Inc., Melville, NY) connected to a Pulnix TM-745E CCD camera (Pulnix Inc., Sunnyvale, CA). A computerized image analysis system, Komet, version 3.1 (Kinetic Imaging Ltd., Liverpool, UK), and Windows, version 3.1 (Microsoft Corporation, Seattle, WA) were used to measure nuclear parameters. Parameters used were tail extent moment (percent DNA in the tail × tail length) and Olive tail moment (percent DNA in the tail × mean tail displacement). Three or more independent experiments were performed for each exposure condition and duplicate slides were made for each exposure. Non-parametric
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Kruskal–Wallis and Mann–Whitney U-tests were used to test for statistical significance among treatments, using the SPSS statistical package (SPSS Inc., Chicago, IL). A P-value of 5 0.05 was considered significant.
2.5. Measurement of cell morphology by hoescht/propidium iodide staining and microscopy Morphological features consistent with apoptosis were assessed through the following modification of the procedure used in Sun et al. (1992), Krown et al. (1996). Following incubations, cells were stained with 0.1 mg/ml hoescht bisbenzimide c 33342 for 5 min, then with 0.5 mg/ml propidium iodide (PI). Cells were scored under fluorescence microscopy (Olympus BX40 System microscope with a BX-FLA fluorescence attachment, DAPI and PI filter cube set) for condensed, pyknotic nuclei and membrane features consistent with apoptosis (Majno and Joris, 1995). Membrane integrity was assessed by PI exclusion, 20–50 cells were scored per treatment. Two or more independent experiments were performed for each treatment and duplicate cultures were made for each exposure.
3. Results
3.1. Cytotoxicity of gossypol after 1 h incubation with and without serum in media Lymphocytes were exposed from 0.01 to 10 mg/ml in media without FBS and from 10 to 80 mg/ml in media with FBS. When lymphocytes were exposed to gossypol without serum in the media, gossypol concentrations of 1 mg/ml or higher were found to cause cytotoxic effects and exposures of 10 mg/ml resulted in 100% loss of viability (Fig. 1). We found that gossypol-induced cytotoxicity was reduced in the presence of serum. For most experiments with serum added, gossypol concentrations of 10–40 mg/ml produced measured viability similar to controls, with ctyotoxic effects seen at 60 mg/ml and above. However, the variability in response to gossypol was greater in media with serum than for serum-free media, and
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in some of these experiments the viability was much reduced.
3.2. DNA strand breaks after 1 h incubation with gossypol with and without serum in media
Viability as percent of control
To assess whether gossypol induced DNA strand breaks in the absence of cytotoxicity, we performed the SCGE assay on cells after 1 h incubations with gossypol. Fig. 2A depicts nuclei from peripheral lymphocytes treated with DMSO only, which exhibited very little DNA fragmentation after electrophoresis. Fig. 2B, in contrast, shows DNA damage in the form of a ‘comet tail’ extending away from the nucleus in cells exposed to 2 mg/ml gossypol. The amount of DNA strand breakage was recorded as the ‘tail extent moment’ (TM), which is the product of the length of the tail and the percent of total DNA from that nucleus that is in the tail region. Table 1A shows the average TM from cells incubated with 0.5, 1.0,
and 2.0 mg/ml gossypol in serum-free media for 1 h. Gossypol was found to increase DNA strand breaks by 7–8 fold over control at concentrations of 2.0 mg/ml, but at this dose cell viability was reduced to less than 50%. Concentrations higher than 2.0 mg/ml caused 100% damaged, unscorable nuclei, and were not tested in the SCGE assay after initial experiments (data not shown). In contrast, H2O2 produced extensive DNA breaks even though cell viability remained high ( \ 95%, Table 1A). We were unable to detect DNA damage caused by gossypol under conditions where cell viability was similar to controls. The production of DNA strand breaks by gossypol was reduced or not seen in the presence of serum proteins. After incubation with gossypol concentrations of 20–80 mg/ml with fetal bovine serum in the media, gossypol induced significant DNA damage only at 80 mg/ml (Table 1B). At this concentration the viability was also affected, being an average of 23%. However, in a few
100%
80%
60%
(-) Series1
(i+) Ser es2
FBS
FBS
40%
20%
0% 0.01
0.1
1
10
100
pol ac C oncentration of go ssy etic aci d ( g/m l) Fig. 1. Cytotoxicity of gossypol at 1 h incubation. Rat lymphocytes were incubated with gossypol at indicated concentrations, both with (left line) and without (right line) FBS in media. Cell viability was assessed by Trypan Blue exclusion (Section 2.1) and is shown as percentage of control (cells incubated with DMSO vehicle control only). Error bars depict standard error of the mean. (FBS, fetal bovine serum, DMSO, dimethylsulfoxide).
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A.
B.
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3.3. DNA strand breaks and cellular morphology after 5 h incubation with gossypol To investigate whether some of the strand breakage observed in earlier studies could be secondary to apoptosis, incubations were carried out for longer times. Serum was included in the media, to preclude apoptosis caused by serum withdrawal. DNA damage was assessed in the SCGE assay and recorded as the Olive tail moment (OliveTM), which is a measure useful for discriminating apoptosis and accidental cell death (Fairbairn et al., 1996). Fig. 3 shows image analysis data of comets from cells exposed to gossypol at Table 1 Induction of cytotoxicity and DNA strand breaks by goosypol in rat lymphocytes (1 h incubation) Treatment Cell viability serum-free (1 h) (mean 9S.D., %)
DNA breaks (TM, mean 9S.D.)c
(a) Treatments in serum free media
Fig. 2. Images of ethidium bromide-stained nuclei from rat lymphocyte cells processed in the comet assay. (A) Undamaged nuclei from cells exposed to DMSO, the vehicle control. (B) Nuclei from cells exposed to gossypol at 2 ug/ml for 1 h as described in Section 2.3. The ‘comet tail’ extends to the positive electrode during electrophoresis. These nuclei exhibit moderate to severe DNA strand breakage; which was quantified by image analysis as described in Section 2.4.
experiments where cell viability was much reduced (two out of nine experiments), we did observe significant DNA damage at lower concentrations of gossypol. For example, in one such experiment, the viability was much lower than average (55, 25, and 18% at concentrations of 20, 40 and 60 mg/ml respectively), and statistically significant increases in TM were seen in the 40 and 60 mg/ml gossypol treatment groups. This confirms that although gossypol can induce DNA damage, the DNA damage was never significantly greater than controls except when accompanied by a reduction in cell viability.
Control DMSO onlyd
99 92
Gossypol 0.5 mg/ml 0.1 mg/ml 2.0 mg/ml
97 90.5 90 9 2 51 9 10b
3 9 17 49 2.4 396.0 239 12.6b
Hydrogen peroxide (H2O2) 30 mM 959 2
165 9 85b
Treatment with serum (1 h)
DNA breaks (TM, mean 9S.D.)c
Cell viability (mean 9S.D., %)
(b) Treatments in media with 10% fetal bo6ine serum Control 98 9 1 1.5 92.8 DMSO onlyd Gossypol 10 mg/ml 40 mg/ml 80 mg/ml
95 9 2 83 925 23 9 39
Hydrogen peroxide (H2O2) 30 mM 89 9 5 a
0.62 91.7 11 922 33933a 839 25b
PB0.5. PB0.0001 (Kruskal–Wallis test followed by Mann–Whitney U-test). c TM, tail extent moment = (comet tail length)×(comet tail percent DNA/100). d DMSO, dimethylsulfoxide. b
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mainly consequent to accidental cell death associated with membrane permeability.
4. Discussion
Fig. 3. DNA fragmentation after a 5 h incubation with gossypol. Rat lymphocytes were incubated with gossypol for 5 h at indicated concentrations in media containing FBS. DNA damage as assessed by the comet assay is plotted with cell viability as measured by Trypan Blue exclusion. DNA damage is recorded as the Olive tail moment (OliveTM) as described in Section 2.4.
20, 40 and 60 mg/ml for 5 h, along with cell viability data. Both the 40 and 60 mg/ml concentrations of gossypol produced a significant increase in OliveTM. However, these concentrations were associated with a significant loss of cell viability, as assessed by Trypan Blue exclusion. The sphingosine-treated cells exhibit a significant increase in DNA damage expressed as a high OliveTM, but little loss of cell membrane permeability (viability \ 70%); a pattern associated with early apoptosis (Majno and Joris, 1995). Assessment of cell and nuclear morphological changes were carried out to confirm that gossypol-induced DNb A strand breaks were not being produced as a result of apoptotic mechanisms. Lymphocytes were exposed 10 – 80 mg/ml gossypol and scored at 1, 3 and 5 h for membrane changes and condensed, pyknotic nuclei associated with apoptosis, using a combination Hoescht/propidium iodide stain. No effect on morphology was seen, although membrane viability was affected as early as 1 h at the high dose (Fig. 4). We therefore, conclude that DNA strand breaks are
Our main finding was that genetic toxicity of gossypol in the SCGE assay is not significant in the absence of cytotoxicity. When cell viability after gossypol treatment was higher than 70% we did not detect a significant increase in DNA strand breaks, either with or without serum in the media. The concentrations of gossypol that induced DNA strand breaks in the presence of cytotoxicity were 2 mg/ml (3.5 mM) and greater without serum in the media and 80 mg/ml and greater when 10% FBS was present in the media (Table 1). The relationship between cytotoxicity and genotoxicity has been the focus of some discussion. In the SCGE assay, necrosis resulting from accidental cell death has been shown to produce fragmented DNA that can mimic genotoxicity or
Fig. 4. Nuclear morphology and viability of rat lymphocytes assessed after 1 and 5 h incubations with gossypol. Rat lymphocytes were incubated with gossypol for 5 h at indicated concentrations in media containing FBS. Condensed, pyknotic nuclei and cell viability were scored as describect in Section 2.5.
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apoptotic damage (Fairbairn and O’Neill, 1995). This observation has led Henderson et al. (1998) suggest that a cut off of 75% viability be used in the SCGE assay to discriminate between ‘true’ genotoxicity and genotoxicity secondary to cytotoxicity. By this criterion, gossypol would not be considered genotoxic in rat lymphocytes as assessed by the work reported here using the SCGE assay. The conclusions from our study are in contrast to the conclusions reached in some previous studies which reported that gossypol induced DNA strand breaks in human fibroblasts and human leukocytes, and concluded that gossypol is genotoxic. The other studies used methods thought to be less sensitive than the SCGE assay (Leroy et al., 1996). Chen et al. (1986) found a dose-dependent increase of DNA single strand breaks as measured by alkaline elution and hydroxylapatite chromatography, when human leukocytes were exposed to 1–15 mg/ml of gossypol for 1 h in serum-free medium. These authors also reported that DNA strand breaks were decreased in cells treated with gossypol in the presence of 10% FCS compared with serum-free media. Nordenskjold and Lambert (1984) had observed DNA single strand breaks using the method of hydroxylapatite chromatography, when human skin fibroblasts were exposed to gossypol at concentrations of 5 –40 mg/ml in serum-free PBS for 30 min at 37°C. Cell viability was not reported. The effect was not seen for a 20 mg/ml exposure in the presence of 2% fetal calf serum. They also measured DNA repair by removing the gossypol after 30 min and following the single strand breaks over time. No repair was seen over 5 h, which is consistent with the DNA breaks being secondary to cytotoxicity. We also explored the possibility that strand breaks might be due to apoptosis by examining DNA damage at further time periods beyond 1 h, as rat spermatocytes showed DNA fragmentation and laddering when exposed to gossypol for 5 h at a concentration of 100 mM (58 mg/ml) and higher (Teng, 1995). Jarvis et al. (1994) also has reported apoptosis-like DNA fragmentation at concentrations of 50–100 mM in HL-60 cells when incubated for 6 h. Shelley et al. (1999) reported DNA
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fragmentation and cell changes consistent with apoptosis in a melanoma cell line exposed to the l-enantiomer of gossypol. We did not find any evidence of apoptosis as assessed by the SCGE assay and cell morphology after a 5 h incubation, and we clearly observed DNA damage from gossypol concomitant with reduced cell viability after a 1 h exposure, which is unlikely to have been due to apoptotic scission (Figs. 3 and 4). It may be that lymphocytes are not susceptible to gossypol-induced apoptosis, unlike other cell types tested, such as cancer cell lines. Teng (1998) also reported reduced viability (67% and lower) at concentrations producing features of apoptosis, so some of the DNA breakage may not have been due to apoptosis but to necrosis. We used lymphocytes as the cell type tested in this study in order to have a primary, untransformed cell type comparable with the cells used in the studies reporting positive results for gossypol-induced DNA strand breaks. A number of theories are postulated to explain the toxic and therapeutic effects of gossypol. Gossypol reacts with many cell macromolecules, binding covalently to epsilon-amino acids through Schiff’s base condensation reactions (Adams et al., 1960). Many of the disruptive effects seen on enzymes and other proteins could be explained by this general mechanism (Strom-Hansen et al., 1989). The many enzymes already known to be inhibited by gossypol (adenylate cyclase, acrosin, protein kinase C, LDH, glutathione-S-transferase, to name a few) have been reviewed by Qian and Wang (1984). Gossypol also chelates iron, copper, aluminum and zinc (Berardi and Goldblatt, 1969); inhibits mitochondrial oxidative phosphorylation (Abou-Donia and Dieckert, 1974); manifests both pro-oxidant and antioxidant characteristics (Coburn et al., 1980; de Peyster et al., 1984; Sheriff et al., 1986; Bender et al., 1988; Janero and Burghardt, 1988; Grankvist, 1989; Laughton et al., 1989), alters membrane potential, fluidity and permeability (Reyes et al., 1984; de Peyster et al., 1986; Benz et al., 1991). and disrupts cell-tocell communication at doses below those necessary to cause appreciable cytotoxicity (Ye et al., 1990; Herve et al., 1996). The vast majority of in vitro studies also report that the presence of
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serum proteins reduces or eliminates the effect of gossypol (see for example Haspel et al., 1984; Hu et al., 1993). Some of the earliest ultrastructural damage occurs in the mitochondrial membrane (Hu et al., 1986; Benz et al., 1990). A study detailing the timing of gossypol-induced toxicity implicates the generation of reactive oxygen species as the primary event (Barhoumi and Burghardt, 1996). Tuszynski and Cossu (1984) and Band et al. (1989) measured cell viability and 3 H-thymidine uptake in cells and observed that effects on DNA synthesis appear after a significant increase in membrane permeability has occurred. This type of evidence suggests that the primary effect of gossypol is interference with one or more non-nuclear cell functions. In summary, a statistically significant increase in DNA strand breaks after exposure to gossypol was demonstrated here. However, DNA strand breaks only occurred in the presence of cytotoxicity, where cell viability was affected. We therefore conclude that gossypol-induced genotoxicity is likely secondary to cytotoxicity in this cell type. This information helps to characterize the safety of gossypol for various clinical uses, as well as to contribute to understanding mechanisms of its toxicity to tumor and non-target cells.
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