Note pubs.acs.org/jnp
Chemopreventive Effects of (−)-Hinokinin against 1,2Dimethylhydrazine-Induced Genotoxicity and Preneoplastic Lesions in Rat Colon Lilian C. Barbosa,† Ricardo A. Furtado,† Humberto C. C. Bertanha,† Iara M. Tomazella,† Eveline S. Costa,† Jairo K. Bastos,‡ Márcio L. Andrade e Silva,† and Denise C. Tavares*,† †
Universidade de Franca, Avenida Dr. Armando Salles de Oliveira 201, 14404-600 Franca, São Paulo, Brazil Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida Café s/n, 14040-903 Ribeirão Preto, São Paulo, Brazil
‡
S Supporting Information *
ABSTRACT: (−)-Hinokinin (1) is a dibenzylbutyrolactone lignan obtained by the partial synthesis of (−)-cubebin. This study reports the antigenotoxic and anticarcinogenic potential of 1 by the comet and aberrant crypt focus assays in the peripheral blood and colon of 4−5-week-old Wistar rats, respectively. The rats were exposed to 1,2-dimethylhydrazine (40 mg/kg) and were treated by gavage with doses of 10, 20, and 40 mg/kg of 1. The results showed that the dose of 40 mg/kg was neither genotoxic nor carcinogenic. In the comet assay, all 1 doses displayed antigenotoxic effects. In addition, this compound (20 and 40 mg/kg) exhibited an anticarcinogenic effect in the aberrant crypt focus assay.
Piper cubeba is a native plant from East India islands, the seeds of which contain (−)-cubebin.1 (−)-Hinokinin (1) is a dibenzylbutyrolactone lignan that can be obtained by the partial synthesis of (−)-cubebin. 1 differs from (−)-cubebin by the presence of a carbonyl moiety at carbon 9 instead of a hydroxyl group.2 In addition, 1 displays trypanocidal3,4 and antimutagenic5,6 activities. In view of the reported biological activities, it was decided to evaluate the antigenotoxic and anticarcinogenic potential of 1. The treatment of the animals did not cause significant weight or water intake changes during the experimental period (Figure 1), indicating the absence of toxicity. The different doses of 1 tested did not increase the extent of DNA damage or aberrant crypt foci, indicating the absence of genotoxic and carcinogenic effects. These findings are in accordance with previous studies from our group showing that 1 was not mutagenic either in vitro5 or in vivo.6 These results are important since the 1 doses tested were the same as those that exhibited in vivo trypanocidal activity.3 Taken together, the absence of genotoxic and carcinogenic effects, under the present experimental conditions, suggests the safe use of this compound. DNA damage was significantly lower in animals treated with the different doses of 1 plus 1,2-dimethylhydrazine (DMH, subcutaneous injection) when compared to the DMH group (Table 1). For carcinogenicity assay, no aberrant crypt foci were observed in the negative control, solvent control, and 1 groups (data not shown). Aberrant crypt foci were observed only in the groups treated with DMH. The analysis of the © XXXX American Chemical Society and American Society of Pharmacognosy
proximal segment showed no significant differences in the number of aberrant crypt foci between animals treated with 1 and DMH and those treated with DMH alone. The anticarcinogenic activity of 1 was observed in the middle and distal segments, where a significantly lower frequency of aberrant crypt foci was observed in the groups treated with 1 plus DMH when compared to the group treated with DMH alone (Table 2). The induction of aberrant crypt foci by DMH Received: February 18, 2014
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DMH was used as carcinogen since it induces DNA damage by various mechanisms. DMH, upon metabolism, exerts a carcinogenic effect by the formation of O6-methylguanine adducts, production of free radicals, and generation of carbonic ions that can methylate DNA, RNA, and proteins.8 Many dibenzylbutyrolactone lignans possess antioxidant activity.9 The protective effect of 1 against DMH-induced DNA damage and aberrant crypt foci observed in this study can therefore be attributed to its ability to scavenge free radicals, acting as an antioxidant agent, since the genotoxic activity of DMH is related to the production of free radicals.8 These results agree with Medola et al.,6 who demonstrated the in vitro antioxidant activity of 1. Furthermore, the antigenotoxic property of 1 has been shown both in vitro5 and in vivo.6 Another mechanism whereby 1 protects against DMHinduced DNA damage and aberrant crypt foci may involve its action on cytochrome P450 enzymes. DMH is metabolized by cytochrome P450 enzymes, and this metabolic activation is necessary for the genotoxicity and carcinogenicity of DMH.10 A successful strategy to prevent DNA damage is the modulation of drug-metabolizing enzymes, such as cytochrome P450, which has been shown to be inhibited by natural products.11 Further development of hinokinin should focus on obtaining new derivatives by adding substituents to the aromatic rings and the benzylic carbon, aiming to improve the activity and selectivity of this drug prototype against other cancer cell and parasite targets. Additionally, the development of formulations using micro- and nanoparticles incorporated with hinokinin is quite important to prevent its biotransformation by the microorganisms in the gastrointestinal tract and to enhance its bioavailability. In conclusion, this study demonstrated that 1 significantly reduces DMH-induced DNA damage and the development of aberrant crypt foci in rats. However, further studies are needed to better understand the molecular mechanisms involved in its chemopreventive effect to allow its safe and effective use in humans.
Figure 1. Body weight gain and water intake of rats treated with 1 and/or DMH and their respective controls in the comet and aberrant crypt foci assays. Negative control, EDTA (vehicle control); Tween 80, 3%; DMH, 1,2-dimethylhydrazine (40 mg/kg of body weight for the comet assay; 160 mg/kg of body weight for the aberrant crypt foci assay); 1, (−)-hinokinin (10, 20, and 40 mg/kg of body weight). Values are the mean ± SD.
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EXPERIMENTAL SECTION
(−)-Cubebin and (−)-Hinokinin. (−)-Cubebin was isolated from the dried seeds of P. cubeba L. as described by Medola.6 (−)-Cubebin was oxidized to 1 as described by Souza et al.3 The identities of (−)-cubebin and 1 (Table S1, Supporting Information) were established by the measurement of its optical rotation, 1H and 13C NMR spectra, and ESIMS. The purities of the compounds were
in a four-week assay was correlated with the development of well-differentiated adenocarcinomas in a 30-week assay.7 In addition, in the present study aberrant crypt foci mainly contained one crypt, being in agreement with literature data for the period of 4 weeks.7
Table 1. Number of Nucleoids Observed in Each Comet Class in Peripheral Blood of Wistar Rats Treated with 1 and/or DMH classg treatment
0 a
negative control Tween 80b 1 (40)f DMHc DMHc + Tween 80b 1 (10)d + DMHc 1 (20)e + DMHc 1 (40)f + DMHc
94.3 95.6 94.5 33.3 40.0 61.6 63.1 76.1
± ± ± ± ± ± ± ±
1 3.1 2.3 2.2 7.0 9.5 10.9 3.3 6.8
5.6 4.5 4.1 59.0 49.1 34.8 33.5 31.5
± ± ± ± ± ± ± ±
2 3.1 2.3 2.6 7.8 16.1 15.8 2.5 6.3
0 0 1.2 7.0 9.8 5.1 3.3 2.3
± ± ± ± ± ±
scoreg
3
1.3 4.9 6.1 3.3 1.0 1.2
0 0 0.1 0.7 1.0 0 0 0.2
± 0.4 ± 1.5 ± 1.7
± 0.7
5.6 4.5 5.0 75.0 72.0 45.0 40.0 37.0
± ± ± ± ± ± ± ±
3.1 2.3 2.7 9.3h 4.8h 14.0h,i 4.0h,i 7.7h,i
% reduction
43.2 50.4 54.7
a Ethylenediamine tetraacetic acid (EDTA; vehicle control). bTween 80 (3%) was used as solvent control. c1,2-Dimethylhydrazine (40 mg/kg of body weight) was used as positive control. d10 mg/kg of (−)-hinokinin/body weight. e20 mg/kg of (−)-hinokinin/body weight. f40 mg/kg of (−)-hinokinin/body weight. gValues are the mean ± SD. hSignificantly different from the negative control (p < 0.05). iSignificantly different from the DMH (p < 0.05).
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Table 2. Number of Aberrant Crypt Foci and Aberrant Crypt Observed in the Proximal, Middle, and Distal Segments of the Colon of Wistar Rats Treated with 1 and/or DMH aberrant crypt focif treatment DMHa DMHa + Tween 80b 1 (10)c + DMHa 1 (20)d + DMHa 1 (40)e + DMHa
proximal 4.5 3.8 1.7 2.7 1.8
± ± ± ± ±
2.2 1.3 1.0 2.0 0.5
aberrant cryptsf
middle 16.2 14.1 10.0 10.0 9.1
± ± ± ± ±
2.9 3.9 5.0 3.9 4.2a
distal 16.5 17.2 12.5 10.2 7.5
± ± ± ± ±
proximal
3.4 4.0 4.6 2.1a 2.8a
5.7 4.3 1.8 3.2 1.8
± ± ± ± ±
2.9 2.1 1.3 2.8 0.5
middle 23.1 20.8 15.8 17.6 14.6
± ± ± ± ±
6.5 7.5 10.1 9.8 8.2a
distal 20.2 23.5 17.3 13.7 10.7
± ± ± ± ±
4.3 6.1 7.8 3.6g 2.9g
a
1,2-Dimethylhydrazine (160 mg/kg of body weight) was used as carcinogen. bTween 80 (3%) was used as solvent control for 1. c10 mg/kg of (−)-hinokinin/body weight. d20 mg/kg of (−)-hinokinin/body weight. e40 mg/kg of (−)-hinokinin/body weight. fValues are the mean ± SD. g Significantly different from the DMH group in the respective column (p < 0.05). described by Senedese et al.13 The stained nucleoids were immediately evaluated at 1000× magnification under a Zeiss fluorescence microscope fitted with a 515−560 nm excitation filter and a 590 nm barrier filter. For each treatment, the extent and distribution of DNA damage in the comet assay were evaluated by examining 100 randomly selected and nonoverlapping nucleoids on the slides per animal. The cells were visually scored and classified according to tail size: class 0, undamaged: no tail; class 1, a short tail whose length was smaller than the diameter of the head (nucleus); class 2, tail length between 1 and 2 times the diameter of the head; and class 3, maximally damaged: a long tail measuring more than twice the diameter of the head. The few comets containing no head and those with almost all DNA in the tail or with a very wide tail were excluded from the analysis. A total of 600 cells were examined per treatment, and the total score was calculated by multiplying the number of damaged cells by the value of the respective comet class and then calculating the sum for each animal. A value of 0 indicated no damage, and a value of 300 indicated maximum damage. The percent reduction in genotoxic agent-induced damage by 1 was calculated as the mean number of cells with damage observed in the treatment with the DNA damage-inducing agent (DMH) minus the number of cells with damage observed in the antigenotoxic treatment × 100, divided by the number of cells with damage observed in the treatment with the DNA damage-inducing agent minus the number of cells with damage in the negative control. Cytotoxicity was evaluated by assessing cell viability using the trypan blue exclusion method. At least 200 cells were counted per treatment.14 Cells were used for the comet assay only when their viability was ≥80%. Aberrant Crypt Foci Assay. After laparotomy, the colons were excised, washed with 0.9% saline, cut open along the longitudinal axis, and divided into three segments (proximal, middle, and distal). The segments were fixed in 10% formalin solution, pH 6.9−7.1, for 24 h. Immediately before analysis, the colons were stained with 0.02% methylene blue for 5 min. Fifty sequential fields were analyzed per colon segment under a light microscope at 100× magnification.7 The number of aberrant crypt foci and crypt multiplicity (number of crypts in each focus) were recorded. Crypt multiplicity is expressed as aberrant crypts/focus. Statistical Analysis. The results were analyzed statistically by analysis of variance (ANOVA) for completely randomized experiments, and F statistics and the respective p-values were calculated. In cases of p < 0.05, treatment means were compared by the Tukey test, calculating the minimum significant difference for α = 0.05. Statistical analysis was performed using the GraphPad Prism 5 program.
estimated to be higher than 97% by both HPLC and spectroscopic analysis. Carcinogen Treatment. The carcinogen DMH (Sigma-Aldrich, lot 112753815004086; purity >99%) was used as an inducer of DNA damage and aberrant crypt foci in the peripheral blood and colon of Wistar rats, respectively. DMH was dissolved in EDTA (37 mg/100 mL distilled water) immediately before use. Animals. Male Wistar rats (Rattus norvegicus) weighing approximately 120 g (4−5 week-old), obtained from the Central Animal House of the University of São Paulo Campus in Ribeirão Preto, were used. The animals were maintained in plastic cages in an experimental room under controlled conditions of temperature (23 ± 2 °C) and humidity (50 ± 10%) on a 12 h light/dark cycle, with ad libitum access to water and commercial diet (Nuvital, PR, Brazil). The study protocol was approved by the Ethics Committee on Animal Use of the University of Franca (Process No. 037/09). General Experimental Procedures. The animals were acclimated for 1 week before the experiment. Each group consisted of six male animals, and the experiments lasted 7 days for the comet assay and 4 weeks for the aberrant crypt foci assay. 1 was dissolved in a solution of water and Tween 80 (97:3) and was administered to the animals by gavage (1 mL/animal) daily for 7 days in the comet assay and five times per week for 4 weeks in the aberrant crypt foci assay. The 1 doses were chosen based on the literature.6,12 The animals were treated with the highest 1 dose tested (40 mg/kg) to evaluate genotoxicity and carcinogenicity by the comet assay in peripheral blood and the aberrant crypt foci assay in the colon, respectively. For assessment of antigenotoxicity and anticarcinogenicity, the animals received different doses of 1 (10, 20, and 40 mg/kg) combined with DMH. The DMH dose and time of treatment in the aberrant crypt foci assay were chosen based on the literature.7 For the comet assay, the animals were injected subcutaneously with 40 mg DMH/kg 4 h before blood collection.13 For the aberrant crypt foci test, a total DMH dose of 160 mg/kg was divided into four subcutaneous injections of 40 mg/ kg, administered in two doses per week for the first 2 weeks. Body weight and water intake were measured daily throughout the experimental period. Comet Assay. All animals were euthanized on day 7, 4 h after treatment with DMH or EDTA (negative control). After laparotomy, the colon was excised, tied at one extremity, and flushed with saline, to remove feces. The other extremity was tied, too, and an enzymatic cocktail (0.3 mg of collagenase I + 5 mg of trypsin/EDTA) was injected into the colon. Next, the colon containing the enzymatic cocktail was placed into phosphate-buffered saline (PBS) and kept in a water bath at 37 °C for 40 min; one colon extremity was cut off to collect the cell suspension. Cell viability was determined on portions of the cell suspension using a dual-dye assay based on a combination of acridine orange and ethidium bromide (under a fluorescent microscope, viable cells that metabolize acridine orange appear green). To accomplish this assay, a 20 μL aliquot of the dye solution was mixed with 20 μL of the cell suspension. Two hundred cells were counted per animal; cells with a viability of ≥80% were used for the comet assay. The slides were prepared, and electrophoresis was performed as
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ASSOCIATED CONTENT
S Supporting Information *
1
H and 13C NMR spectra of 1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (D. C. Tavares). Tel: +55 16 3711 8871. Fax: +55 16 3711 8878. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by São Paulo Research Foundation (FAPESP; grant no. 2008/02211-0), Brazil. REFERENCES
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