EFFECTS OF LONG-TERM TREATMENT OF COLON ADENOCARCINOMA WITH CROCIN, A CAROTENOID FROM SAFFRON (CROCUS SATIVUS L.): AN EXPERIMENT

Abstract: We used an experimental model in the rat to examine the effects of long-term treatment with crocin, a glycosylated carotenoid from the stigmas of the saffron crocus, on colon cancer. BD-IX rats were divided into four groups: Groups G1 and G2, designated "cancer groups, "were used to study the effects of crocin on the progression of colon cancer, and Groups G3 and G4, designated "toxicity groups," were used to study the effects of the treatment on metabolic processes and the parenchyma. DHD/K12-PROb cells were injected subcutaneously into the chest of Group G1 and G2 animals. From 1 to 13 weeks after inoculation, animals in Groups G2 and G4 received a weekly injection of crocin (400 mg/kg body wt sc). Animals in Groups G1 and G3 received no treatment. In addition, lines of animal and human colon adenocarcinoma cells (DHD/K12-PROb and HT-29) were used to perform assays in vitro to examine the cytotoxicity of crocin.

Life span was extended and tumor growth was slower in crocin-treated female rats, but no significant antitumor effect was found in male rats. Acute tubular necrosis was found in all kidney samples from crocin-treated animals, but slight signs of nephrotoxicity were found by biochemical analysis of the serum. In assays in vitro, crocin had a potent cytotoxic effect on human and animal adenocarcinoma cells (HT-29 and DHD/K12-PROb cells, 50% lethal dose = 0.4 and 1.0 mM, respectively). Treated cells exhibited a remarkable loss of cytoplasm and wide cytoplasmic vacuole-like areas. In conclusion, long-term treatment with crocin enhances survival selectively in female rats with colon cancer without major toxic effects. The effects of crocin might be related to its strong cytotoxic effect on cultured tumor cells.

Introduction
The need for anticancer drags with high efficacy and low toxicity has led to studies of putatively protective factors in fruits, vegetables, herbs, and spices. Favorable results have been obtained with many natural compounds in recent years, suggesting the possible utility of further investigations.

Dietary factors, such as spices, are attractive sources of antitumor agents. Saffron, which consists of the dry stigmas of the plant Crocus sativus L., is used as a spice and a food colorant. In folk medicine, it has been used in the treatment of numerous diseases, including tumors. In recent years, the antitumor properties of crude extracts of saffron have been demonstrated in vitro and in vivo ( 1-5), and it is of great interest that nontumor cells in culture appear to be less sensitive or even insensitive to the effects of such extracts compared with tumor cells ( 1, 5).

Chemical analysis of saffron extracts has revealed that characteristic components include the carotenoids crocin and crocetin as well as the monoterpene aldehydes picocrocin and safranal ( 6, 7). The cytotoxic activity of saffron carotenoids was demonstrated with cells from a variety of murine and human tumors, and although individual cell lines varied in terms of sensitivity, both carotenoids always had cytotoxic effects at similar respective concentrations ( 5, 8). Moreover, normal untransformed cells appeared to be almost insensitive to the carotenoids from saffron in vitro ( 2, 4, 5). In an earlier study using human cervical epitheloid carcinoma (HeLa) cells, we compared the cytotoxic activities of each component of saffron and found that crocin, the glycosylated carotenoid, had a major inhibitory effect ( 9). Morphological observations of treated cells suggested the induction of apoptosis by crocin ( 9).

Few studies have been performed in vivo to examine the antitumor activity of saffron carotenoids. Nair and co-workers ( 4, 5) reported a considerable increase in the life span of tumor-bearing mice, with tumors of various origins, when animals were treated with dimethylcrocetin, a deglycosylated derivative of crocin. Details of the effects of crocin have not been elucidated. Crocins are unusual, in that they are water-soluble carotenoids, as a consequence of their glycosylated state, so they are easy to administer. Thus they appear to be the most appropriate components of saffron for evaluation as potential anticancer agents.

The aim of this study was to examine the antitumor effects and the toxicity of long-term treatment with crocin in an experimental model of colon adenocarcinoma in the rat and to examine the cytotoxic effects of crocin on tumor cells in vitro.

Materials and Methods
Cell Culture
We used DHD/K12-PROb (also called DHD/K12-TRb) cells for assays in vivo and in vitro. This clonal cell line was initially established by Martin and colleagues ( 10) in 1983 from a colon adenocarcinoma that had been induced in BD-IX rats by administration of 1,2-dimethylhydrazine. DHD/ K12-PROb and HT-29 cells (a line of human colon adenocarcinoma cells) were cultured as monolayers in a 1:1 (vol/ vol) mixture of Dulbecco's modified Eagle's medium and Ham's F-10 medium (GIBCO BRL Life Technologies, Paisley, Scotland) supplemented with 10% fetal bovine serum (GIBCO BRL) and gentamicin (0.005%; GIBCO BRL). Cells were passaged after dispersion in 0.125% trypsin in EDTA.

Assay of Cytotoxicity In Vitro
Assays of cytotoxicity in vitro were performed using DHD/K12-PROb and HT-29 cells. Cells at the exponential phase of growth were washed twice with phosphate-buffered saline (PBS), trypsinized, and resuspended in complete culture medium at 4.0 x 104 cells/ml. Cells were plated at 8 x 103 cells/well in 96-well flat-bottomed tissue culture plates and allowed to adhere to wells during an overnight incubation at 37degreesC. Lyophilized purified crocin was dissolved in sterilized water and diluted to appropriate concentrations with culture medium. Unattached cells were removed from dishes with PBS, and the solution of crocin in culture medium was added to wells to give a total volume of 100 mul. All samples were tested in duplicate. Cells were incubated for 24 hours, and then 10 mul of a 5 mg/ml solution of 3-[ 4, 5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide in PBS were added to each well. After a four-hour incubation at 37degreesC, the resultant formazan precipitate was solubilized in 100 mul of dimethyl sulfoxide, and absorbance was measured at 540 nm with a multiwell plate reader (model MR 5000, Dynatech, Burlington, MA).

Staining of Cells
DHD/K12-PROb cells with or without prior treatment with crocin were stained by a modified version of the Wright-Giemsa fast-staining method, as described in the manual from the manufacturer (Grifols, Barcelona, Spain).

Animals
Male and female BD-IX rats were used. They were taken from a colony established at the authors' animal facility from founders that had been purchased from a commercial breeder (CRIFFA, Barcelona, Spain). Animals were maintained in compliance with European Community Directive 86/609/CEE for the use of laboratory animals. As recommended by the Federation of European Laboratory Animal Science Associations, rats in the animal facility are tested annually to ensure that the colony remains free of pathogens such as Mycoplasma pulmonis, Salmonella spp., Sendai virus, Hantaan virus, and Toolan H1 virus.

From birth to the end of the experiments, all rats were kept in protective cages with unlimited access to water and standard rat chow (Panlab, Barcelona, Spain). At the beginning of the experiments, rats were six weeks old and weighed 100-150 g.

Implantation of Tumors and Design of the Experiment
A total of 48 animals were used. Each animal was assigned randomly to one of four groups, with male and female rats being distributed equally in each group. Groups were defined as follows: Group G1, cancer control group (n = 16); Group G2, crocin-treated cancer group (n = 12); Group G3, toxicity control group (n = 10); Group G4, crocin-treated toxicity group (n = 10).

In animals in the so-called cancer groups (G1 and G2), tumors were generated in the thoracic region by unilateral subcutaneous injection of DHD/K12-PROb cells into the right side of the chest. DHD/K12-PROb cells were trypsinized, washed, and resuspended in PBS. Then 0.25 ml of the suspension, containing 1 x 106 cells, was injected into each rat.

From 1 to 13 weeks after inoculation, animals in Group G2 received a subcutaneous injection of crocin (400 mg/kg body wt), into the caudal third of the back, once a week. For injection, lyophilized crocin was dissolved in 1-2 ml of sterile saline solution to give a solution of 45-60 mg/ml. The solution was passed through a 0.22-mum-pore filter before injection. Animals in Group G1 received no treatment.

Tumor growth was monitored in all animals in the cancer groups and recorded weekly by measurement of greatest diameters with calipers.

In the case of the two toxicity groups (G3 and G4), the treatment protocol was identical. However, because these groups were used to study the effects of crocin on metabolic processes and in the parenchyma, no animals were inoculated with tumor cells.

The body weights of all animals were measured weekly. Rats in both cancer groups were not sacrificed, and on the day each rat died a natural death was recorded. Animals in the toxicity groups were sacrificed during the first week after the end of treatment, and blood and samples of lung, liver, kidney, and spleen were collected as follows. Rats were anesthetized with an intraperitoneal injection of a mixture of ketamine (75 mg/kg) and xylazine (10 mg/kg), and approximately 1.5-2.0 ml of blood were withdrawn from each rat by cardiac puncture. A lethal dose of thiopental sodium was then given intracardially. Finally, animals were autopsied, and samples from all lobules of the liver, the spleen, the kidneys, and all lobules of the lungs were taken. All samples of tissue were processed for histopathological examination.

Extraction of Crocin
Dry stigmas of pure "La Mancha" saffron (Crocus sativus L.) were purchased locally in Albacete, Spain, and stored in darkness at 4degreesC before use.

Crocin was purified by mild acid hydrolysis of saffron dry stigma, adsorption chromatography, and high-performance liquid chromatography, as described by Escribano and co-workers ( 9).

Microscopic Examination of Tissue
Tissue samples were fixed in formalin for 48 hours and then embedded in paraffin. Sections (4 mum) were cut and stained with hematoxylin-eosin by standard procedures.

Biochemical Analysis of Serum
Blood samples were processed for biochemical analysis, and levels of the following factors in the serum were measured: glucose, urea, creatinine, calcium, sodium, potassium, chloride, cholesterol, triglycerides, total bilirubin, aspartate aminotransferase, alanine aminotransferase, L-gamma-glutamyltransferase, alkaline phosphatase, cholinesterase, lactate dehydrogenase, amylase, total protein, and albumin.

Biochemical analyses were performed by autoanalyzers (model 747, Hitachi) following the methods described in the manual of the reagent manufacturer (Boehringer Mannheim, Mannheim, Germany). Specifically, these methods were as follows: glucose, cholesterol, triglycerides, L-gamma-glutamyltransferase, and amylase serum levels were measured by enzymatic colorimetric tests, urea by kinetic ultraviolet test, creatinine by the Jaffe method (kinetic) without deproteinization, calcium by o-cresolphthalein complexion, sodium, potassium, and chloride by selective electrode with dilution, total bilirubin by the DPD method, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase by ultraviolet tests, alkaline phosphatase by an optimized standard method, total protein by the biuret reaction, and albumin by bromcresol green.

Statistical Analysis
We used the Mantel-Cox test to compare the survival time in crocin-treated and nontreated animals and Student's t-test to compare tumor size and body weight in each group. Analysis of variance was used for comparison of biochemical data in the toxicity groups.

Statistical significance was recognized at p < 0.05.

Results
Figure 1 shows the cytotoxic effects of crocin on the two different lines of colon adenocarcinoma cells. A dose-dependent cytotoxic effect was observed. The 50% lethal doses of crocin were estimated to be 0.4 and 1.0 mM for HT-29 and DHD/K12-PROb cells, respectively. The cytotoxic effects of 2 mM crocin on DHD/K12-PROb cells were examined under the light microscope after cells were stained by a modified version of the Wright-Giemsa method. We observed a marked loss of cytoplasm and wide cytoplasmic vacuole-like areas in such cells (Figure 2).

Among rats with tumors, female rats treated with crocin lived significantly longer than others (p = 0.04) (Figure 3). Furthermore, external diameters of tumors were smaller in treated female rats from seven weeks after inoculation to the end of the experiment (Figure 4).

Although the duration of survival was also increased by crocin in the male rats, the difference was not significant for male rats in the two cancer groups (Figures 3 and 4).

No significant differences in body weight were observed at any time during the experiment.

Results of the biochemical analysis of serum from the toxicity groups are shown in Table 1. Compared with the toxicity control group (G3), crocin-treated female rats had significantly elevated serum levels of calcium and sodium. There were also slight increases in levels of creatinine. By contrast, the level of potassium was reduced (Table 1). Among male rats, crocin-treated animals had significantly elevated serum levels of calcium, cholesterol, cholinesterase, and total protein and reduced levels of urea, aspartate aminotransferase, and alkaline phosphatase. There were also slight increases in levels of albumin (Table 1). In male and female rats, serum glucose levels were significantly lower than in nontreated animals.

Histological examination revealed acute tubular necrosis in all samples of kidney from crocin-treated animals (Figure 5). Such lesions were mainly found in the proximal convoluted tubes, in which epithelial necrosis and desquamation were observed. Tubular lumens contained necrotic cells and amorphous debris. In a few cases, eosinophilic material filled Bowman's space, collapsing glomerular tufts. The extent of necrotic lesions was defined as mild, moderate, or severe. Acute tubular necrosis was severe in four male and two female rats, moderate in two female rats, and mild in one male and one female rat. According to the classification of the World Health Organization, these lesions were classified as toxic acute tubular necrosis.

Samples of kidney from nontreated animals show no alteration.

No specific changes were observed in samples of lung, liver, and spleen.

Discussion
In this study, we observed the potent cytotoxic effects of crocin on lines of human and animal adenocarcinoma cells (HT-29 and DHD/K12-PROb, 50% lethal dose = 0.4 and 1.0 mM, respectively; Figure 1). In previous studies, cytotoxic activity of crocin was investigated using different tumor cell lines, with concentrations needed to produce 50% cytotoxicity of 2-19 mug/ml ( 5, 9). The present study reveals that the cytotoxic activity of crocin on colon adenocarcinoma cells is lower than that observed on carcinoma, sarcoma, and leukemia tumor cells. The differences in sensitivity between different cell types could be due to the existence of distinct cell surface receptors, intracellular retention transport, or differences in the drug uptake. Adenocarcinoma cells treated with crocin exhibited wide cytoplasmic vacuole-like areas and remarkable loss of cytoplasm, but their nuclei remained apparently unchanged (Figure 2).

Using an experimental model of colon adenocarcinoma, we also examined the effects of long-term treatment with crocin on the life span of rats and on tumor growth. Crocin increased the survival time and decreased tumor growth in female rats, without any significant similar effects in male animals. Nair and colleagues ( 4) showed that saffron ethanolic extracts significantly increased the life span of mice with different types of tumors. The reported increase in survival (45-120%, depending on the tumor model) was much greater than that observed in our study with the purified carotenoid (11.9% and 12.4% in male and female rats, respectively; Figure 3). Because of the different characteristics of tumor models, animal species, cell lines, drug doses, and administration methods (oral vs. subcutaneous injection), comparison of the above-mentioned data is not plausible.

To our knowledge, the present work analyzes for the first time the antitumor activity of purified crocin, the major glycosylated water-soluble carotenoid. We chose to focus on a model of colon adenocarcinoma in the rat because of the relatively slow growth of such tumors, which reflects the time course of many human tumors and, in addition, because colon cancer is one of the most frequent human neoplasms. The cell line used in the present study was established from a colon adenocarcinoma. The aggressiveness of the tumor generated by these cells in BD-IX rats and the development of lung metastases have been well documented ( 12, 13). Our results also showed that, in treated female rats, tumors were smaller than in nontreated animals during the longest duration of treatment. Although this difference was not statistically significant, it might suggest a relationship between survival time and tumor size.

Thus, after weekly administrations of a relatively high dose of crocin, we found that crocin appears to have antitumor activity in vivo, supporting results of present and previous assays in vitro ( 9).

Although we used a relatively high dose of crocin, longterm treatment did not result in severe deleterious metabolic changes in rats. No data about toxic effects of crocin have been reported, so our study is the first to investigate such effects. Glucose serum levels were lower in treated than in control animals. This result agrees with that reported by El Daly and associates ( 14), who observed a greater decrease in blood glucose of rats treated with C. sativus stigmas and cisplatin than in those treated with cisplatin alone. The mechanism of this alteration remains unknown, but it could be related to an increase of insulin levels mediated by pancreatic dysfunction. Anatomic-pathological lesions in kidney induced by crocin were diagnosed as toxic acute tubular necrosis, but no major manifestations in the biochemical analyses of serum were found, only a slight increase of calcium levels, and the electrolyte imbalance in female rats could be related to this process. Although the mechanism of crocin nephrotoxicity is unclear, the normal levels of creatinine found in blood might indicate that tubular damage induced by the drug has a reversible clinical course. On the other hand, small variations in serum transaminase activities in male rats might suggest only slight hepatotoxicity. Nonetheless, it is possible that other therapeutic schedules might have less toxic effects without loss of potential benefits, and further pharmacological studies are needed to determine the optimum protocol for administration of crocin for antitumor therapy.

On the other hand, the selective action of crocin in female rats compared with male rats suggests that the effects of crocin in animals might be related to hormonal factors.

In conclusion, long-term treatment with crocin enhanced survival in female rats with colon cancer selectively, without major toxic effects. Thus crocin appears to have antitumor activity in vivo and might play a major role in the antitumor effects of saffron. Further studies are needed to assess the value and possible application of crocin in cancer chemotherapy.

Acknowledgments and Notes
This study was supported in part by "Plan Nacional de I +/- D" (Spain) Grant SAF97-0149. J. Ontanon is the recipient of a fellowship from the Cultural Albacete Public Consortium. Address reprint requests to Dr. Damian Garcia-Olmo, Unidad de Investigacion, Hospital General de Albacete, Hnos. Falco s/n, 02006 Albacete, Spain. FAX: 34-967 24 39 52. E-mail: dgolmo@arrakis.es.

Submitted 8 April 1999; accepted in final form 16 July 1999.

Table 1. Serum Levels of Biochemical Parameters in Male and Female Rats in the Toxicity Groups (Control and Crocin-Treated)[a]

Legend for Chart:

A - Biochemical Factor
B - Male Rats: Group, Control
C - Male Rats: Group, Crocin-treated
D - Male Rats, P value
E - Female Rats: Group, Control
F - Female Rats: Group, Crocin-treated
G - Female Rats, P value

A B C
D E
F G

Glucose, mg/dl 350.8 +/- 61.7 223.4 +/- 34.5
0.006 234.0 +/- 39.4
172.0 +/- 40.2 0.02

Urea, mg/dl 41.0 +/- 4.0 34.2 +/- 4.6
0.04 39.4 +/- 6.4
40.2 +/- 4.5 NS

Creatinine, mg/dl 0.6 +/- 0.0 0.6 +/- 0.0
NS 0.6 +/- 0.0
0.7 +/- 0.1 NS

Calcium, mg/dl 9.9 +/- 0.5 10.6 +/- 0.4
0.04 10.7 +/- 0.4
11.6 +/- 0.5 0.01

Sodium, meq/l 139.2 +/- 1.8 143.4 +/- 5.7
NS 139.0 +/- 2.0
144.6 +/- 4.6 0.05

Potassium, meq/l 5.1 +/- 0.7 5.0 +/- 0.5
NS 4.6 +/- 0.2
4.4 +/- 0.1 0.03

Chloride, meq/l 98.8 +/- 2.9 101.2 +/- 5.9
NS 99.2 +/- 2.3
103.0 +/- 5.9 NS

Cholesterol, mg/dl 53.0 +/- 6.4 63.0 +/- 3.4
0.02 65.6 +/- 16.1
77.4 +/- 18.9 NS

Triglycerides, mg/dl 281.6 +/- 126.5 293.2 +/- 89.4
NS 192.6 +/- 55.6
328.2 +/- -175.7 NS

Total bilirubin, mg/dl 0.10 +/- 0.0 0.05 +/-0.1
NS 0.12
+/-0.0 0.05
+/-0.1 NS

Aspartateaminotransferase,
U/l 137.4 +/- 26.1 103.3 +/- 13.0
0.04 89.4 +/- 17.9
109.3 +/- 30.1 NS

Alanineaminotransferase,
U/l 45.8 +/- 3.8 47.8 +/- 9.9
NS 44.6 +/- 3.8
59.5 +/- 22.3 NS

L-chi-glutamyltransferase,
U/l 8.4 +/- 8.6 0.3 +/- 0.5
NS 3.0 +/- 7.4
0.3 +/- 0.5 NS

Alkaline phosphatase, U/l 356.8 +/- 6.1 308.0 +/- 27.6
0.03 223.2 +/- 46.2
231.8 +/- 74.5 NS

Cholinesterase, U/l 107.7 +/- 5.5 144.4 +/- 20.7
0.01 645.0 +/- 852.8
1,810.2 +/- 580.4 NS

Lactate dehydrogenase,
U/l 1,910.3 +/- 473.3 1,226.3 +/- 370.4
NS 875.6 +/- 476.2
1,379.8 +/- 513.2 NS

Amylase, U/l 6,349.3 +/- 604.3 6,100.3 +/- 589.9
NS 5,804.0 +/- 467.4
6,390.3 +/- 691.0 NS

Total protein, g/dl 6.0 +/- 0.4 6.7 +/- 0.4
0.04 7.4 +/- 0.9
7.8 +/- 1.0 NS

Albumin, g/dl 3.1 +/- 0.2 3.5 +/- 0.3
NS 3.9 +/- 0.4
4.3 +/- 0.6 NS
a: Values are means +/- SE. NS, not significant.

GRAPH: Figure 1. Dose-dependent inhibition of proliferation of 2 lines of adenocarcinoma cells, HT-29 and DHD/K 12-PROb, by crocin. Cells were incubated with crocin at indicated concentrations for 24 h before assessment of viability.

PHOTOS (BLACK & WHITE): Figure 2. Control DHD/K12-PROb cells (A) and DHD/K12-PROb cells that had been incubated for 24 h with 2 mM crocin (B). Modified Wright-Giemsa staining. Magnification x200.

GRAPH: Figure 3. Survival of male and female rats in 2 cancer groups.

GRAPH: Figure 3. Survival of male and female rats in 2 cancer groups.

GRAPH: Figure 4. Time courses of growth of tumors in male and female rats.

PHOTOS (BLACK & WHITE): Figure 5. Light-microscopic images of kidney samples from control and crocin-treated animals. A: unaltered renal parenchyma in a control rat. B: tubular necrosis in a crocin-treated rat. Magnification x200.

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By Dolores C. Garcia-Olmo; Hans H. Riese; Julio Escribano; Jesus Ontanon; Jose A. Fernandez; Manuel Atienzar and Damian Garcia-Olmo

D.C. Garcia-Olmo, J. Ontanon, M. Atienzar, and D. Garcia-Olmo are affiliated with the Experimental Research Unit, Albacete General Hospital, 02006 Albacete, Spain. H. H. Riese is affiliated with the Immunology and Oncology Department, National Center of Biotechnology, 28049 Madrid, Spain. J. Escribano is affiliated with the School of Medicine and J. A. Fernandez with the Biotechnology Division, Institute for Regional Development, University of Castilla-La Mancha, 02071 Albacete, Spain.

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