http://www.paper.edu.cn Toxicology in Vitro 16 (2002) 41–46 www.elsevier.com/locate/toxinvit Photo-induced cytotoxicity of malonic acid [C60]fullerene derivatives and its mechanism X.L. Yang*,C.H. Fan,H.S. Zhu Research Center of Materials Science, Beijing Institute of Technology, PO Box 327, Beijing 100081, PR China Accepted 10 August 2001 Abstract The biological activities of fullerenes have attracted extensive attention in recent years. The aim of this paper is to study the relation of the photo-induced cytotoxicity of fullerene derivatives to their chemical structures as well as the possible cellular mechanism involved in the photocytotoxicity. Three C60 derivatives with two to four malonic acid groups (DMA C60,TMA C 60 and QMA C60) were prepared and the cytotoxicity of these compounds against HeLa cells was determined by MTT. Cell cycle was measured by flow cytometry. The results showed that the cytotoxicity of the malonic acid C60 derivatives was irradiation- and dose- dependent. The sequence of their photo-induced growth inhibition was DMA C60>TMA C60 >QMA C60. Hydroxyl radical quencher mannitol (10 mm) was not able to prevent cells from the damage induced by irradiated DMA C60. DMA C60,together with irradiation,was found to have an ability of inducing a decrease in the number of G 1 cells from 63 to 42% and a rise in that of G2+M cells from 6 to 26%. These data indicated that the number of malonic acid molecules added to C60 played an important role in the phototoxicity,and the blockage of cell cycle might be a mechanism of this activity. # 2002 Published by Elsevier Science Ltd. Keywords: Cell cycle; Cell growth; Malonic acid C60 derivatives; Photo-induced; Reactive oxygen species 1. Introduction 1996; Schuster et al.,1996; Tsuchiya et al.,1996). Among various kinds of C60 derivatives,the malonic In recent years,aqueous solutions of C 60 and its deri- acid adducts of C60,first synthesised by Lamparth and vatives have been prepared by various methods to study co-workers (Hirsch et al.,1994; Lamparth and Hirsch, their biological effects (Yang et al.,1998). Some pro- 1994),may have very potent applications in the biome- gress has been made in their activities against enzymes dical field,due not only to their unique physical and (Ando et al.,1993; Fridman et al.,1993; Sijbesma et al., chemical properties,but also to their simple massive 1993; Schinazi et al.,1994; Nacsa et al.,1997; Iwata et preparation. These properties included: (i) minor mod- al.,1998),DNA molecules (Tokuyama et al.,1993; ification in the structure of parent C60 cage; (ii) good Boutorine et al.,1994; An et al.,1996) and free radicals solubility and poor aggregation formation in aqueous (Nagano et al.,1994; Chiang et al.,1995; Sun et al., solution when the number of addends is bigger than 1997; Cheng et al.,2000a). There have been a few one,and this aggregation will lead to the significant papers concerning the influence of C60 and its deriva- reduction of the lifetime of the excited triplet state tives on cell growth as well (Tokuyama et al.,1993; Li et (Guldi and Asmus,1999); (iii) reliable availability of a al.,1994; Schinazi et al.,1994; Scrivens et al.,1994; variety of regioisomers with defined three-dimensional Tsuchiya et al.,1995; Irie et al.,1996; Nakajima et al., structures (Lamparth and Hirsch,1994). Therefore, many recent papers have dealt with the biological con- Abbreviations: DMA C60,dimalonic acid C 60; MTT,methylthiazo- cerns of the malonic acid adducts of C60 (Okuda et al., lyldiphenyl-tetrazolium bromide; OD,optical density; QMA C 60, 1996,2000; Dugan et al.,1997; Satoh et al.,1997a,b; quadrimalonic acid C60; ROS,reactive oxygen species; TMA C 60,tri- Huang et al.,1998; Guldi and Asmus,1999; Mashino et malonic acid C60 * Corresponding author. Tel.: +8610-6891-1949; fax: +8610-6891- al.,1999). For example,two regioisomers with C 3 or D3 5023. symmetry,containing three malonic acid groups per E-mail address: [email protected] (X.L. Yang). molecule,were found to be neuroprotective in mice, 0887-2333/02/$ - see front matter # 2002 Published by Elsevier Science Ltd. PII: S0887-2333(01)00102-3 中国科技论文在线 http://www.paper.edu.cn 42 X.L. Yang et al. / Toxicology in Vitro 16 (2002) 41–46 both in vitro and in vivo and able to prevent apoptosis 2.3. Preparation of aqueous solutions of C60 derivatives in human hepatoma cells (Dugan et al.,1997; Huang et al.,1998). However,the photo-induced cytotoxicity of The stock solutions of DMA C60,TMA C 60 and malonic acid derivatives of C60 and the related QMA C60 were prepared in pH8 buffer (0.2 m mechanism have not yet been examined. In this pre- NaH2PO4/Na2HPO4). Sterile solutions for cell experi- liminary study,three malonic acid derivatives of C 60 ments were obtained by filtering these solutions with 0.2 were chosen to investigate their effects on the growth mm pore membranes. and cell cycle of human cervix uteri tumor-derived HeLa cells. They were dimalonic acid C60 (DMA C60), 2.4. Cytotoxicity determination trimalonic acid C60 (TMA C60) and quadrimalonic acid C60 (QMA C60),the structures of which are shown in Human cervix uteri tumor-derived HeLa Cells were Fig. 1. Our results showed their cytotoxicity was irra- cultured in an atmosphere with 5 CO2 and at 37 C pro- diation dependent and decreased with the number of vided by a NAPCO CO2 incubator in RMPI 1640 med- malonic acid molecules added to C60. It was also indi- ium containing 10% heat-inactivated fetal calf serum and cated cell-cycle blockage is possibly related to the 100 U/ml penicillin and 100 mg/ml streptomycin. photo-induced cytotoxic activity of the fullerene com- Cytotoxicity determination of C60 derivatives was pound. carried out with 24-well plates. Four repeats were undertaken for each sample. When cells were attached to the bottom of culture 2. Materials and methods plates,the culture medium was replaced by the fresh medium containing 2% fetal calf serum and C60 deriva- 2.1. Chemicals and technical equipment tives or/and mannitol. Then the cultures were irradiated with a 300 W halogen lamp set at 30 cm over the cul- C60 (99% purity) was prepared as previously reported tures. To avoid heat damage to the cells,the heat pro- (Cheng et al.,2000a). RMPI 1640 medium,fetal calf duced by the lamp was removed by a piece of heat- serum,MTT reagent and propidium iodide were pur- shield glass. Irradiation was carried out three times, chased from Sigma Chemical Co. The NAPCO CO2 each time for 0.5 h with a 24-h interval between each incubator and the MODEL 550 microplate reader were irradiation. After the third irradiation,the viable cell products of Precision Scientific and Bio-Rad,Inc., number was determined immediately by MTT assay respectively. Cell-cycle analysis was carried out with a (Mosmann,1983; Zhu et al.,2000). MTT reagent can be FACS 420 flow cytometer (Becton Dickinson Co.). converted into blue formazan only by viable cells. Lysis of cells produced a blue solution,the optical density 2.2. Synthesis of malonic acid derivatives of C60 value (OD value) of which was measured with a MODEL 550 microplate reader in a dual wavelength, Malonic acid derivatives of C60 were synthesised as with the measurement wavelength at 450 nm and the described in our previous paper (Cheng et al.,2000a). reference wavelength at 655 nm. The data obtained were Briefly,C 60 and diethyl bromomalonate was mixed at a statistically analyzed using Student’s t-test. ratio of 1:6 at room temperature under an Ar atmo- Growth inhibition was represented as (1Àmean OD sphere,so three malonate derivatives of C 60 with two to value of sample/mean OD value of control)Â100%. four additional groups could be produced simulta- neously and then separated macroscopically by silica-gel 2.5. Cell-cycle analysis chromatography. Hydrolysis with NaH and CH3OH led to the formation of the corresponding acids. Flow cytometry was used to analyze the cell cycle according to the method described previously (Zhu et al.,2000). Briefly,cells with or without treatment of DMA C60 or/and irradiation were collected by cen- trifuge (1000 rpm/10 min) after trypsinization and then fixed in 75% ethanol overnight at 4 C. Fixed cells were stained with propidium iodide to show the DNA content of each cell. The strength of the fluo- rescence within a single cell was detected under a FACS 420 flow cytometer. 100,000 cells were counted in total for each sample. The percent of cells in differ- Fig. 1. The malonic acid derivatives of C60 used for cytotoxic investi- ent phases (G1,S and G 2+M) were obtained by the gation in the present experiment. They included dimalonic acid C60 (DMA C60),trimalonic acid C 60 (TMA C60) and quadrimalonic acid utilization of the ‘Multicycle’ software attached to the C60 (QMA C60). flow cytometer. 中国科技论文在线 http://www.paper.edu.cn X.L. Yang et al. / Toxicology in Vitro 16 (2002) 41–46 43 3. Results (63%)>TMA C60 (55%)>QMA C60 (31%),suggesting that the increase of side group number in C60 cage may 3.1. Photo-induced cytotoxicity of malonic acid C60 reduce its photo-induced activity. The photo-induced cytotoxicity of dimalonic acid C60 3.2. Influence of hydroxyl radical quencher mannitol on (DMA C60) is shown in Fig. 2. Without irradiation, photo-induced cytotoxicity of DMA C60 DMA C60 in the range of 0–64 mm did not produce any toxicity against HeLa cells.