Toxicology 159 (2001) 135–141

Cytotoxicity of derivatives from dehydrocrotonin on V79cells and Escherichia coli

Patricia da Silva Melo a,*, Nelson Duran b, Marcela Haun a

a Department of Biochemistry, Institute of Biology, State Uni6ersity of Campinas (UNICAMP), CP 6110,Campinas SP 13083-970, Brazil

b Biological Chemistry Laboratory, Institute of Chemistry, State Uni6ersity of Campinas, CP 6154,Campinas SP 13081-970, Brazil

Received 16 September 2000; accepted 7 November 2000

Abstract

New derivatives from dehydrocrotonin (DHC, compound I), with the same anti-ulcerogenic properties but lesstoxicity were synthesised by reducing the cyclohexenone moiety of DHC with NaBH4 (compound II), by reducing thecyclohexenone and lactone moieties with LiAlH4 (compound III) and by transforming the lactone moiety into anamide (compound IV) using dimethylamine. The cytotoxicity of these derivatives from DHC was assayed on V79fibroblast cell line. Three independent endpoints for cytotoxicity were evaluated; namely, the nucleic acid content(NAC), tetrazolium reduction (MTT) and neutral red uptake (NRU). IC50 values of 540 and 350 mM were obtainedfor compound II in the NRU and NAC tests, respectively. Compound III was less toxic than the other DHCderivatives (IC50=1800 mM) on V79 cells based on NAC assay. Compound IV showed an IC50 ranging from 350 to600 mM based on the three endpoints evaluated. The three compounds were less toxic on V79 cells than DHC. DHC,compounds II, III and IV did not change the respiration rate of Escherichia coli on the acute toxicity assay. © 2001Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Dehydrocrotonin; Cytotoxicity; V79 fibroblasts; MTT; Neutral red

www.elsevier.com/locate/toxicol

1. Introduction

Different endpoints have been used to assesscytotoxicity in vitro as alternatives for toxicityusing animals, including the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium

bromide (MTT), neutral red uptake (NRU) andnucleic acid content (NAC) (De Conti et al.,1998). These endpoints generally assess differentaspects of cellular functions. The reduction ofMTT assesses the functional intactness of mito-chondria based on the enzymatic reduction of atetrazolium salt by the mitochondrial dehydroge-nase of viable cells (Denizot and Lang, 1986;Loveland et al., 1992). NRU is a measure oflysosomal integrity since it reflects the capacity of

* Corresponding author. Tel.: +55-19-37887886; fax: +55-19-37887840.

E-mail address: marcela@reitoria.unicamp.br (P. da SilvaMelo).

0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0300 -483X(00 )00417 -0

P. da Sil6a Melo et al. / Toxicology 159 (2001) 135–141136

viable cells to incorporate vital dye into theseorganelles (Renzi et al., 1993; Reppetto and Sanz,1993). Finally, assay of nucleic acid content evalu-ates the total cellular material as indication oftotal cell number (Cingi et al., 1991; Haun et al.,1992).

Croton cajucara (sacaca) is a medicinal plantfound in the Amazon and is used by local nativesfor its gastroprotective properties. Earlier studiesshowed that the trans-dehydrocrotonin (DHC),the major secondary metabolite present in thebark of Sacaca, has anti-ulcerogenic activity (Ro-driguez, 1998; Souza-Brito et al., 1998) and anti-tumour activity (Grynberg et al., 1999). However,Rodriguez and Haun (1999) showed that DHChas basal and specific cytotoxic effects on V79cells and hepatocytes.

In an attempt to expand our knowledge of thetoxic effects of DHC, we studied the cytotoxicityeffects of DHC derivatives on V79 fibroblast cul-tures comparing the structure-toxicityrelationship.

2. Materials and methods

2.1. DHC and deri6ati6es

DHC was obtained from C. cajucara (Sacaca)barks as described by Souza-Brito et al. (1998).The lactone DHC molecule was opened through adimethylamine reaction to give compound IV, asdescribed by Cromwell and Cook (1958); 0.5 g ofDHC was dissolved in 2.0 ml of MeOH and 3.0ml of an aqueous 25% solution of dimethylaminewas heated in a sealed tube at 75°C for 72 h. Thereaction mixture was evaporated to dryness andthe product then purified by thin layer chro-matography to yield compound IV (Fig. 1) as ayellow oil. The reduction of ketone group in DHCwas done as described by Itokawa et al. (1989)using a methanol solution of DHC (100 mg)treated with an excess of sodium borohydride(NaBH4, 30 mg). After work-up in the usual way,the product was purified by thin layer chromatog-raphy (n-hexane-EtOAc-MeOH, 7:2:1 v/v) andafforded compound II (Fig. 1) as a white powder(Itokawa et al., 1989). For compound III, DHC

(300 mg) in 50 ml of tetrahydrofuran (THF) wasadded to a solution of LiAlH4 (130 mg) in THF.The mixture was refluxed with stirring for 2 h andworked up as usual to give a crude crystallinematerial, which was separated by thin layer chro-matography (EtOAc-hexane 1:1, v/v) and yieldeda white powder (Shimoma et al., 1998). The pu-rity and structure of compounds II–IV was char-acterised by NMR, UV, IR and MS techniques.

2.2. V79 culture

The cytotoxic effect of DHC derivatives, ex-pressed as cell viability, was assessed on a perma-nent lung fibroblast cell line derived from chinesehamsters (V79), which is commonly used for cyto-toxicity studies (Souza-Brito et al., 1998; Ro-driguez and Haun, 1999; Aoyama et al., 2000).V79 fibroblasts were grown as mono layers inDulbecco’s modification of Eagle’s medium(DMEM), supplemented with 10% heat-inacti-vated fetal calf serum (FCS), 100 IU/ml penicillinand 100 mg/ml streptomycin in a humidified incu-bator with 5% CO2 in air at 37°C. Cells wereplated at density of 3×104/ml in 96-well plates.The medium was removed 48 h after cell seedingand replaced with medium containing DHCderivatives at concentrations ranging from 80 to650 mM. The substance was first dissolved inmethanol and then in DMEM. The final concen-tration of methanol in the test medium and con-trols was 1%. Cells were exposed for 24 h to testmedium with or without DHC derivatives (con-trol). Each drug concentration was tested in fourreplicates and repeated three times in separateexperiments. At the end of incubation, three inde-pendent endpoints for cytotoxicity were evalu-ated; MTT, NAC and NRU.

2.2.1. NACCell number in control and treated wells was

estimated from their total nucleic acid contentaccording to Cingi et al. (1991). Cells were washedtwice with cold phosphate buffered saline (PBS)and a soluble nucleotide pool was extracted withcold ethanol. The cell monolayers were then dis-solved in 0.5 M NaOH at 37°C/1 h. The ab-sorbance at 260 nm of the NaOH fraction was

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used as an index of cell number (Bianchi andFortunat, 1990). Results are expressed as meanpercentage of absorbance at 260 nm in treated wellscompared with controls.

2.2.2. MTTThe tetrazolium reduction assay was performed

according to the method of Denizot and Lang(1986). Briefly, cells were washed once with PBSbefore adding 0.1-ml serum-free medium contain-ing MTT (1mg/ml) to each well. After incubationfor 4 h, the supernatant was removed and the blueformazan product obtained was dissolved in 1 mlethanol with stirring for 15 min on a microtitre plateshaker and the absorbance was read at 570 nm.

2.2.3. NRUThe NRU assay was performed according to the

method of Borefreund and Puerner (1984). After 4 hof incubation with serum-free medium containing50 mg neutral red/ml, the cells were washed quicklywith PBS and then 0.1 ml of a solution of 1% (v/v)acetic acid: 50% (v/v) ethanol was added to eachwell to extract the dye. After rapid agitation on amicrotitre plate shaker the absorbance was read at540 nm.

2.2.4. In 6itro bacterial toxicity assayThe toxicity of the DHC and derivatives was

evaluated in comparison to the toxicity of tetracy-cline by measuring the inhibition of the respira-

Fig. 1. Dehydrocrotonin and derivatives

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tion of Escherichia coli cultures. The toxicity testis described in detail elsewhere (Jardim et al.,1990). Basically, the assay comprises the incuba-tion of E. coli cultures at 37°C with knownamounts of the stressing agent. When the CO2

concentration produced by microbial respirationreached 0.5 mmol/l, or approximately 9×108 cell/ml, 45 ml of the E. coli culture was transferredinto several flasks and each one received 5 ml ofone sample withdrawn at a selected concentrationof the DHC or derivatives (final concentration 1mM). As a positive control, tetracycline was usedat a concentration of 1 mM, which inhibits 50% ofthe respiration bacterial. As a negative control, 5ml of distilled water was introduced in one of theflasks and the CO2 production monitored every20-min using flow injection analysis (FIA). Thetoxicity test was followed for a maximum periodof 120 min. For incubation periods of more than120 min, there is loss of CO2 to the atmospheredue to CO2 over saturation (\5 mmol/l) in theaqueous culture medium. The bacteria (ATCC25922) used in the in vitro bacterial toxicity assaywas provided by the microbiological laboratory ofthe UNICAMP hospital.

2.2.5. Statistical analysisExperiments were done three times (four repli-

cates each) in separate experiments. To calculatethe IC50 values (concentration that produces a50% inhibitory effect on the evaluated parameter),the results were transformed to percentage ofcontrols and the IC50 were graphically obtainedfrom the dose-response curves. Results are ex-pressed as the mean9S.D.

3. Results

The viability assays which measure the ‘killingcapacity’ or the ‘metabolic capacity’ of the testmaterials was applied with a broad range of con-centrations, as is usual when starting to examinethe toxicity of unknown compounds (Cingi et al.,1991). The cytotoxic effects of the compounds II,III and IV were measured by MTT, NAC andNRU assays on V79 cells. The results indicatedthat V79 cell viability after 24-h exposure was

Fig. 2. Viability of V79 cells after treatment with compound IIfor 24 h. Endpoints evaluated; neutral red uptake (NRU),nucleic acid content (NAC) and MTT reduction. Each pointrepresents the mean9S.D. of three experiments in four repli-cates.

sufficient to produce the dose-dependent effectsshown in Figs. 2 and 4, causing detachment andloss of cells at the effective doses. The IC50 valueswere obtained mathematically from the concen-tration-response curves.

Compound II showed higher toxicity on NAC(IC50=350 mM) than the NRU (IC50=500 mM)and apparently no toxicity on V79 cells evaluatedby MTT reduction until a concentration of 500mM (Fig. 2), indicating a stimulus on MTT reduc-tion. Fig. 3 shows that the compound III was lesstoxic on V79 cells evaluated by the three viabilityassays than compounds II, IV and DHC. TheIC50 value obtained from the concentration-re-sponse curve was 1800 mM evaluated throughnucleic acid content. Compound III did not showsignificant toxicity on V79 cells evaluated eitherthrough MTT reduction and NRU, as there was80% viability until 2.5 mM.

Compound IV showed similar toxicity evalu-ated by NAC and MTT reduction (IC50=500 and

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600 mM, respectively). The IC50 value obtainedfrom the curve was 350 mM evaluated throughNRU (Fig. 4).

Fig. 5 shows that DHC and derivatives did notinhibit the respiration of E. coli in the majorconcentrations used (1 mM).

4. Discussion

A balance between the therapeutic versus toxi-cological effects of a compound is an importantparameter when verifying its applicability as apharmacological drug. Cell culture can be used toevaluate basal cytotoxicity (Ekwall and Ekwall,1988) and target organ toxicity (Balls and Fen-tem, 1992; Seibert et al., 1996) and in some cases,may provide information on the lethal dose invivo (Shrivastava et al., 1991). In this study, V79fibroblasts were used because this cell line is well

Fig. 4. Viability of V79 cells after treatment with compoundIV for 24 h. Endpoints evaluated; neutral red uptake (NRU),nucleic acid content (NAC) and MTT reduction. Each pointrepresents the mean9S.D. of three experiments in four repli-cates.

Fig. 3. Viability of V79 cells after treatment with compoundIII for 24 h. Endpoints evaluated; neutral red uptake (NRU),nucleic acid content (NAC) and MTT reduction. Each pointrepresents the mean9S.D. of three experiments in four repli-cates.

characterised and commonly used in mutagenicityand toxicity studies (Cingi et al., 1991). E. coli(ATCC 25922 strain) was used for determinationof bacterial cytotoxicity by FIA proved to be areliable and rapid assessment of the toxicity of thecompounds by analysing the alteration of theamount of CO2 produced and trapped in theculture medium (Jardim et al., 1990). In this as-say, DHC and derivatives did not show any toxiceffect on E. coli assay. Some authors have sug-gested that MTT reduction is a measurement ofmitochondrial function, in particular of succinicdehydrogenase activity. However, detailed studieswith liver homogenate fractions and intact hepa-tocytes indicated that hepatocyte MTT reductionis predominantly an assessment of cytosolicNAD(P)/NAD(P)H redox balance (Fry et al.,1995). The presence of inhibitor or inducers ofreactions from the redox balance confirms theinvolvement of it on MTT reduction capacity for

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V79 cells. This stimulus in MTT reduction ob-served on cells treated with compound II may beexpected as a result of cytosolic NAD(P)H levels.It has been recently postulated that superoxideanion is capable of reducing tetrazolium com-pounds such as MTT (Andrews et al., 1997; Ro-driguez and Haun, 1999).

Compounds II, III and IV showed lower toxic-ity than the DHC, the parent compound, whichshows IC50 ranging between 230 and 360 mM. Inthis way the modifications in the DHC changedthe toxicity of the DHC derivatives, althoughcompound III lost the anti-ulcerogenic activity(data not show). The compounds (DHC, II, IIIand IV) were assayed in three models of gastriculcers (HCl/ethanol, stress and indomethacin in-duced) in mice according to Hiruma-Lima et al.(1999). Compound II did not loose its anti-ulcero-genic activity, its toxicity was less than that ofDHC, probably because of the loss of the hexe-nenone moiety such as in a similar manner inAflatoxicol, pentenone moiety reduced from Afla-toxin B1. Aflatoxicol is 18 times less toxic thanAflatoxin B1 evaluated by histopathological assay(Detroy and Hesseltine, 1970).

According to Giordano et al. (1992), the struc-ture-activity relationship of sesquiterpene lactonesprovides experimental and theoretical evidencethat the presence of a non-sterically hindered

Michael acceptor seems to be an essential struc-tural requirement for the cytoprotective activity ofthese compounds. These data corroborate ourresults where compound IV, despite that changein the lactone moiety, retained its anti-ulcerogeniceffects because of the presence of a Michael ac-ceptor substitute (C(O)N(CH3)2). On the otherhand, compound III would have lost the anti-ul-cerogenic activities because the absence of aMichael acceptor and, probably, the toxic andpharmacological effects of sesquiterpene lactonescould be due to the presence of this kind ofacceptors.

In summary, the use of different endpoint testsshowed that the compounds II, III and IV dif-fered in toxicity compared with DHC and providesome evidence of the mechanism of toxicity ofsesquiterpene lactones.

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