In vivo evaluation of the mutagenic potential and phytochemical characterization of oleoresin from Copaifera duckei

We characterized the chemical constituents of Copaifera duckei oleoresin and used dermal application to Wistar rats to evaluated its possible mutagenic and cytotoxic activities on peripheral blood reticulocytes and bone marrow cells. Chemical characterization of the oleoresin revealed the presence of sesquiterpene hydrocarbons, an unidentified neutral diterpene and diterpene acids. To evaluate mutagenicity evaluation the rats were treated with 10, 25 and 50% of the LD50 dose of the oleoresin for three consecutive days and peripheral blood collected after 0, 24, 48 and 72 h for micronucleus analysis. The rats were humanly sacrificed 24 hours after the last treatment and chromosome preparations made using standard techniques. At the three concentrations and the three time intervals tested we found that there were no statistically significant differences in either the mean number of micronucleated reticulocytes (MNRETs) or the number of chromosomal aberrations as to the negative control. However, at 25 and 50% of the LD50 dose of the oleoresin there was a significant decrease in the mitotic index (MI) as compared to the negative control. Under our experimental conditions, C. duckei V11 oleoresin produced no mutagenic effects on bone marrow cells or in peripheral reticulocytes as assessed by chromosome aberrations and the micronucleus test respectively, but showed cytotoxic activity at high doses.


Introduction
The oleoresin obtained by tapping the trunk of trees of the genus Copaifera (Caesalpinaceae), is widely used in Brazilian popular medicine under the name 'óleo de copaíba' (copaiba oleoresin), predominantly as a healing, antiseptic and anti-inflammatory agent (Le Cointe, 1934;Pio Corrêa, 1984).
Copaiba oleoresins have been used as unique vegetal drugs despite the existence of more than 20 species of Copaifera in Brazil (Dwyer, 1951) and the significant inter and intra species differences in chemical composition (Cascon and Gilbert, 2000) copaiba oleoresins have been used medicinally throughout Brazil.The oleoresin is a natural solution of diterpene acids in an essential oil composed mainly of sesquiterpenes and has been reported as being bactericidal (Maruzzela and Sicurella, 1960;Opdyke, 1976;Cascon et al., 2000;Tincusi et al., 2002), anti-helminthic (Pellegrino, 1967;Gilbert et al., 1972), analgesic (Fernandes and Pereira, 1989), anti-inflammatory (Basile et al., 1988;Fernandes et al., 1992;Veiga-Junior et al., 2001) and gastro-protective (Paiva et al., 1998) as well as showing antitumor (Ohsaki et al., 1994;Lima et al., 1998) and trypanocidal (Cascon et al., 1998) activity.However, in several of these evaluations commercial copaiba oleoresins were used, the chemical composition of which was either not given or only partially described.
There exists considerable interest in determining the risks that plant extracts may pose to health, since many of these extracts contain compounds known to cause diseases or even death to animals and humans by acting as natural mutagens and carcinogens (Panigrahi and Rao, 1982;Araújo et al., 1999;Burim et al., 1999;Chacon et al., 2002).The objective of the study described in this paper was to characterize the chemical constituents of Copaifera duckei oleoresin and evaluate its mutagenic and cytotoxic potential by applying the micronucleus test to peripheral blood and analyzing chromosomal aberrations in bone marrow cells of Wistar rats treated with this oleoresin.

Plant material and chemical analysis
We collected 4.4 litres of Copaifera duckei Dwyer (V11) oleoresin from trees growing at a site in Mazagão county in the Brazilian state of Amapá near the town of Macapá at 00°02'56" N; 051°44'46" W on the 7 of December 1996.The collection of oleoresin and botanical material was made by Vera Cascon and Jonas de Oliveira Cardoso with the collaboration of the Amapá Institute of Scientific and Technological research (Instituto de Pesquisas Científicas e Tecnológicas do Estado do Amapá, IEPA).Botanical identification was made by Antônio Sérgio Lima da Silva, Museu Paraense Emílio Goeldi (MG), Belém, Pará, Brazil.The botanical material collection number was 031 deposited at 12/02/1998.
An equal volume of dichloromethane was added to the crude oleoresin which was esterified with diazomethane in ether and analyzed using gas chromatography -mass spectrometry (GC-MS) in a Hewlett Packard HP 6890 chromatograph (column 30 m x 250 mm x 0,25 mm) -HP 5 mass spectrometer (70 eV, mass selective detector 5972 A), using PFK as a reference.The temperature was started at 70 °C, rising by 2 °C per minute to 300 °C.
Both sesquiterpenes and methyl esters of diterpene acids were analyzed in the same sample and the majority of the compounds were characterized using the Wiley Library/ Mass Spectra 275 and by comparison of retention times with data published by Braga (1994).

Animals and assay procedures
Experiments were carried out using six-week-old Wistar rats (Rattus norvegicus) weighing 90-110 g acquired from Alfenas University animal house and kept in polyethylene boxes (n = 6) in a climate-controlled environment (25 ± 4 °C, 55 ± 5% humidity) with a 12h light/dark cycle (07:00h to 19:00h) and fed Labina-Purina (Agribrands Purina do Brasil Ltda, Paulínia, São Paulo, Brazil) and water ad libitum.The rats were divided into three experimental and two control groups each containing three females (F 1 to F 3 ) and three males (M 1 to M 3 ).Rats in the experimental groups received 10%, 25% or 50% of the LD 50 dose (7.467 mg/kg body weight, Carvalho and Cascon, 2003) of Copaifera duckei oleoresin by dorsal dermal injection for 3 consecutive days at 24 h intervals.The negative control group received 0.9% (w/v) NaCl by the same route as the experimental rats and the positive control group 30 mg of cyclophosphamide/kg body weight.
For the micronucleus test blood smears were collected using peripheral tail blood from experimental and control rats, the blood being collected before the first injection (0 h) and at 24, 48 and 72 h after the first injection (Hayashi et al., 1990).A total of 8000 reticulocytes were analyzed per rat, 2000 for each collection time.All rats were humanly sacrificed 72 h after the first injection, each rat being injected intraperitoneally with 0.5 mL of 0.16% (w/v) aqueous colchicine 90 min prior to euthanasia.Bone marrow was obtained at autopsy (t = 72 h) for the analysis of chromosome aberrations in metaphase cells using the method of Ford and Hamerton (1956).The UNIFENAS Animal Bioethical Committee approved the present study on 17 th August 2003.
To detect micronuclei and chromosome aberrations slides were Giemsa stained and 100 metaphases per animal analyzed to determine the mean number of chromosomal aberrations in a blind test.Chromosomal aberrations were classified according to Savage (1976) as gaps, breaks, deletions, fragments, rings and dicentric chromosomes.Gaps were recorded but not included in the statistical analysis.The mitotic index was obtained by counting the number of mitotic cells in 1000 cells per animal.The data were submitted to one-way analysis of variance (ANOVA) and the Tukey-Kramer multiple comparison test using the GraphPad Instat ® software version 3.01 (GraphPad Software, Inc., San Diego, USA).Results were considered statistically significant at p < 0.05.

Mutagenic and cytotoxic evaluation
The results obtained in the in vivo test system are presented in Tables 1 and 2. The micronuclei assay showed no 834 Maistro et al. statistically significant differences in the mean number of micronuclei (MN) in peripheral blood reticulocytes (RETs) of the rats in any of the experimental groups as compared between themselves or with the negative control group (Table 1).At the three concentrations tested, a small but statistically non significant increase was observed between the mean number of micronucleated reticulocytes (MNRETs) after 24, 48 and 72 h as compared with their respective 0 h controls.No sex differences were observed between any of the groups.
As compared to rats in the negative control group, the mitotic index (Table 2) of rats in the 10% LD 50 group was not significantly different but rats in the 25% and 50% LD 50 groups showed a significant decreases (p < 0.05 and p < 0.01 respectively).
There were no statistically significant differences in the mean number of chromosome aberrations between the three experimental groups and the negative control group (Table 2).In all treatments with Copaifera oleoresin the most frequent chromosomal aberrations observed were chromatid breaks, followed by chromatid gaps, deletions and isochromatidic gaps.

Discussion
The in vivo rat micronuclei test and chromosome aberrations assay are two of the most frequently used and sen-sitive tests for investigating the genotoxic profile of chemicals, these tests having been recommended for routine analysis because they produce results that are considered highly relevant in the human context (Morita et al., 1997;Preston et al., 1987).The Copaifera duckei oleoresin analyzed by us was very rich in diterpene acids and possessed moderate amounts of sesquiterpene hydrocarbons, confirming the report by Cascon and Gilbert (2000) that there are differences in the chemical composition of the oleoresin produced by different Copaifera species.
Terpenes are abundant in superior plants and show a shared structure of isoprene units, the sesquiterpenes (C 15 H 24 ) having three such units and the diterpenes (C 20 H 32 ) four (Robbers et al., 1997).Some sesquiterpenes and diterpenes are known to be cytotoxic and to inhibit tumors, with toxic sesquiterpenes generally containing one or more functional alkylating groups which suggests that they are possibly mutagenic and carcinogenic (Cassady and Baird, 1990;Wall et al., 1998).
Our data shows that treatment C. duckei oleoresin resulted in depression of mitotic activity and no statistically significant increase in chromosome aberrations in bone marrow cells and in the mean number of MNRETs in the peripheral blood of Wistar rats.The dose related decrease in mitotic index ( oleoresin depresses mitosis at high doses.Although not statistically significant, the dose related increase in the mean number of MNRETs observed at the two higher doses probably occurred due to cumulative effects of the resin because the rats were treated during three consecutive days. Sena and Chen (1998) used the micronucleated cell assay and Swiss albino mice to evaluated the in vivo bone marrow cell mutagenic potential of 25, 50 and 80% of the LD 50 dose of orally administered Copaifera langsdorfii oleoresin and demonstrated a statistically significant increase micronucleated cells (and hence mutagenic action) 836 Maistro et al.only at high doses, although these authors used a higher maximum dose than we did.Donaldson et al. (1994) extensively investigated the cytotoxicity of the antimitotic antitumor diterpene taxol derived from the yew tree Taxus brevifolia and showed that the antimitotic effect of taxol classified it as an important anticancer agent.A genotoxicity study by Dias et al. (1997) showed that taxol had no radio-sensitizing effect on chromosomal aberrations induced by gamma radiation and also did not increase doxorubicin-induced chromosomal aberrations in in vitro Chinese hamster ovary cells.
The active component of Eremanthus elaeagnus wood oil is the sesquiterpene eremanthine, genotoxic evaluation in vivo in rodents and in vitro in human lymphocytes having showed that low concentrations of eremanthine produced no cytotoxic or clastogenic effects and that only doses of 400 mg Kg -1 showed toxicity (Dias et al., 1995).Another sesquiterpene, Glaucolide B, isolated from Vernonia eremophila produced no significant increase in the frequency of chromosomal aberrations in mouse bone marrow cells but showed cytotoxic and clastogenic effects on human lymphocytes in vitro, indicating that caution is needed in its medicinal use (Burim et al., 1999).
Available information about the evaluation of the mutagenic potential of copaiba oleoresin in different rodent species has shown that despite some qualitative differences between the Copaifera oleoresins studied the toxic pattern was similar, with cytotoxic and some genotoxic effects only occurring at high doses.Since there are about 20 different Copaifera species in Brazil and the oleoresins obtained from these plants have been used in popular medicine, it is important to establish the relationship between chemical composition and biological activity of authentic samples of the oleoresins in order to permit their validation as safe and effective phyto-medicines and to allow adequate quality control.
The results of our study demonstrate that under the experimental conditions employed Copaifera duckei oleoresin presented cytotoxic effects at high doses but did not induce a statistically significant increase in the mean number of chromosome aberrations in the bone marrow cells or in the mean number of MNRETs from the peripheral blood of Wistar rats in vivo.

Table 1 -
Number of micronucleated reticulocytes (MNRETs) observed in the peripheral blood cells of female (F 1 to F 3 ) and male (M 1 to M 3 ) Wistar rats treated with Copaifera duckei oleoresin.For each time period (0, 24, 48, 72 h) 2000 cells were analyzed, giving a total of 8000 cells per animal.
*Significantly different from negative control and all other treatments at p < 0.001.SE = standard error.

Table 2 -
Mitotic Index and distribution of the different types of chromosomal aberrations (CA) observed in bone marrow cells of female (F 1 to F 3 ) and male (M 1 to M 3 ) Wistar rats treated with a Copaifera duckei oleoresin.For each treatment 100 cells per animal were analyzed, giving a total of n = 600 cells per treatment.