Services on Demand
Print version ISSN 0001-3765
On-line version ISSN 1678-2690
An. Acad. Bras. Ciênc. vol.76 no.1 Rio de Janeiro Mar. 2004
Cleverson Busso; Marialba A. A. Castro-Prado
Universidade Estadual de Maringá, Departamento de Biologia Celular e Genética Av. Colombo 5790, 87020-900 Maringá, PR, Brasil
Cremophor EL is a solubilizer and emulsifier agent used in the pharmaceutical and foodstuff industries. The solvent is the principal constituent of paclitaxel's clinical formulation vehicle. Since mitotic recombination plays a crucial role in multistep carcinogenesis, the study of the recombinagenic potential of chemical compounds is of the utmost importance. In our research genotoxicity of cremophor EL has been studied by using an uvsH//uvsH diploid strain of Aspergillus nidulans. Since it spends a great part of its cell cycle in the G2period, this fungus is a special screening system for the study of mitotic recombination induced by chemical substances. Homozygotization Indexes (HI) for paba and bi markers from heterozygous B211//A837 diploid strain were determined for the evaluation of the recombinagenic effect of cremophor EL. It has been shown that cremophor EL induces increase in mitotic crossing-over events at nontoxic concentrations (0.05 and 0.075% v/v).
Key words: Aspergillus nidulans, mitotic recombination, cremophor EL, Homozygotization Index, antineoplasm agents.
Cremofor EL (CEL) é um solubilizante e emulsificante amplamente utilizado nas indústrias farmacêuticas e de gêneros alimentícios. É o principal veículo empregado nas formulações clínicas do antineoplásico paclitaxel. Considerando-se que a recombinação mitótica desempenha importante função no processo de carcinogênese, o estudo de substâncias químicas com potencial recombinagênico assume importância crucial, no sentido de se detectar aquelas que eventualmente possam atuar como promotoras de neoplasias. A genotoxicidade do cremofor EL foi estudada no presente trabalho, utilizando-se uma linhagem diplóide uvsH//uvsH de Aspergillus nidulans. Neste fungo as células vegetativas comumente repousam no período G2 do ciclo celular, facilitando a ocorrência da recombinação mitótica. O efeito recombinagênico do CEL foi avaliado através da determinação dos Índices de Homozigotização para os marcadores nutricionais paba e bi do diplóide heterozigoto B211//A837. Os resultados demonstram que CEL é efetivo em induzir crossing-over mitótico em concentrações não tóxicas ao fungo (0.05 e 0.075% v/v).
Palavras-chave: Aspergillus nidulans, recombinação mitótica, cremofor EL, índices de homozigotização, agentes antineoplásicos.
The organic solvent cremophor EL (CEL) (polyoxyethyleneglycerol triricinoleate 35) is a viscous liquid produced by the reaction of castor oil (Ricinus communis) with ethylene oxide (Hoffman 1984). CEL is employed as a vehicle for the solubilization of a wide variety of hydrophobic drugs, including anesthetics, photosensitizers, sedatives, immunosuppressive agents and anticancer drugs (Gelderblom et al. 2001).
Without causing serious toxicity, in vitro cremophor EL reverts the Multidrug Resistance (MDR) phenotype in clinically executable concentrations. Multidrug resistance is a mechanism by which cancer cells are able to survive diverse drugs in structurally unrelated groups. Overexpression of the multidrug transporter protein, also known as P-glycoprotein, has often been observed in MDR cells. This transmembrane protein is capable of pumping a wide variety of chemotherapeutic agents out of the cells, protecting them from the agent's toxic effects (Breier et al. 2000). Cremophor EL affects the metabolism of MDR cells, alters cell membrane properties, and impairs the P-glycoprotein function. Actually the solvent favors the action of antineoplasm drugs, such as paclitaxel, doxorubicin and vinblastine (Fjällskog et al. 1993, Friche et al. 1993, Woodcock et al. 1990).
The cytotoxic effect of CEL, found in tumoral human cells resistant to doxorubicin, has been associated with peroxidation of polyunsaturated fatty acids and with direct disturbing effect in the cell membrane (Nygren et al. 1995, Burton 1991, Bégin et al. 1988).
The transformation of normal human cells into cancer cells is a multistep process, while mitotic recombination is a mechanism involved in bringing about such transformation (Nowell 1976, Barrett 1993). In heterozygous cells bearing a mutant and normal alleles for a tumor suppressor gene, the somatic recombination may turn up to be a promoter of neoplasms by inducing homozygosis of the mutant tumor suppressor allele (Maher et al. 1993, Sengstag 1994).
Mitotic crossing-over has already been recorded in Drosophila melanogaster, Aspergillus nidulans, Saccharomyces cerevisiae and mammalian cells in culture, including human cells. It is currently thought to be a common occurrence process in diploid cells (Ramel et al. 1996, De La Torre et al. 1994, Maher et al. 1993, Kunz et al. 1981, Stern 1936).
The filamentous fungus A. nidulans is an excellent organism for studying mitotic crossing-over. This is chiefly due to two important factors: a) A. nidulans's mitosis has many common characteristics with higher eukaryotes mitosis, and b) the fungus spends most of its vegetative cell cycle in G2 phase. At this phase, the presence of two copies of each chromosome favors the occurrence of mitotic crossing-over (Bergen and Morris 1983, Iwanejko et al. 1996).
Since several reports have suggested somatic recombination in mechanisms leading to carcinogenesis and due to the fact that CEL is used as a solubilizer of hydrophobic drugs, such as antineoplasm agents, we decided to examine the ability of this solvent to induce mitotic recombination.
MATERIALS AND METHODS
The genotypes and origin of A. nidulans strains are provided in Table I. Diploid strain (B211//A837) (Busso et al. 2001) was prepared according to Roper (1952).
Complete (CM) and minimum medium (MM) were prepared according to Van de Vate and Jansen (1978). Selective medium was prepared with MM and nutritional requirements of each strain. Solid medium was prepared with 1.5% agar; incubation for strain growth was done at 37ºC.
EVALUATION OF DRUG TOXICITY
Conidia of diploid strain B211//A837 were inoculated in the plate center with MM + pyridoxine (control) and MM + pyridoxine + cremophor EL (0.05% and 0.075% v/v). Nine plates incubated at 37C were used for each dose and for control. Diameters of colonies were measured after 24, 48, 72, 96 and 120 hours of incubation. Student's t test compared colony diameters with and without the drug.
Colonies of B211 strain were cultivated in dialysis membranes placed aseptically on the surface of petri dishes with CM and CM + 0.05% and 0.075% v/v of CEL. Incubation occurred for 30 h, at 37ºC, and samples taken during 4-28 h period. Membranes were stained with cotton-blue-lactophenol and analyzed under an optic microscope.
EVALUATION OF THE RECOMBINAGENIC POTENTIAL (PIRES AND ZUCCHI 1994)
Conidia of the diploid strain were inoculated in MM + pyridoxine + cremophor EL (0.05% and 0.075% v/v) and incubated for 5 days at 37ºC. Treatment produced visible diploid sectors, D1-D6, identified by their different morphology from the original diploid. Diploid sectors were submitted to spontaneous haploidization in CM after purification in MM + pyridoxine. Only haploid segregants were selected for recombinagenesis test (Franzoni et al. 1997). Conidia of each haploid sector were transferred to 25 defined positions in CM plates (master plates). After 48 hours of incubation at 37ºC, colonies were transferred to selective media and the phenotypical analysis of the haploid segregants was carried out.
The treatment with CEL in MM + pyridoxine produces only heterozygous (+//- or -//+) or homozygous (+//+) segregants since the recessive ones (-//-) fail to grow in MM + pyridoxine (Table II). After haploidization of diploids D1-D6 the nutritional markers will segregate among the haploids in the proportion of 4+: 4-, if solvent fails to induce recombinagenesis; or 4+: 2-, if solvent induces crossing-over. Values of Homozigotization Indexes (HI) (the ratio between number of prototrophic and auxotrophic segregants) equal or above 2.0 evidence the recombinagenic effects of the substance under analysis (Pires and Zucchi 1994, Chiuchetta and Castro-Prado 2002a,b). Results were compared by Yates corrected Chi-square test.
HIs for genes paba and bi were determined to evaluate diploid B211//A837 after treatment with cremophor EL.
RESULTS AND DISCUSSION
Aspergillus nidulans reproduces itself asexually by forming multicellular conidiophores and uninucleate spores called conidia. We have first studied the effects of CEL on mycelia growth of B211//A837 strain and on conidiophore morphology of B211 strain. Solvent was added to a pyridoxine-supplemented minimal agar medium to obtain final concentrations of 0.05% and 0.075% v/v. Both CEL concentrations had no effect on mycelia growth or on conidiophore morphology (results not shown). Only a slight delay in the timing for conidiophore formation of B211 strain was observed (Table III).
In this study, the B211//A837 diploid strain, homozygous for uvsH mutation, was used as a sensitive system for the evaluation of the genotoxic activity of CEL in nontoxic concentrations.
Various repair mechanisms are mobilized to restore the original DNA sequence when DNA damages occur. These mechanisms include base excision repair (BER), nucleotide excision repair (NER), mutagenic repair and post-replication repair (Goldman et al. 2002, Hjertvik et al. 1998, Fishel and Kolodner 1995). In A. nidulans the UV-sensitive mutants (uvs) have been classified in different epistatic groups such as: UvsB, UvsC, UvsF and UvsI. The study of mitotic intergenic recombination in this filamentous fungus may be greatly facilitated by the use of UvsF group mutations: gene mutation uvsH, that operates in the post-replication repair pathway, is responsible for high frequencies of mitotic intergenic recombination in homozygous condition (Osman et al. 1993, Iwanejko et al. 1996).
It has been shown that CEL induces a statistically significant increase in mitotic crossing-over events in A. nidulans. The treatment of B211//A837 with CEL increased Homozygotization Indexes for paba and bi markers (Tables IV and V). This effect, nevertheless, was not dose-dependent.
It is believed that mitotic recombination involving heterozygous cells for a deleterious gene triggers carcinogenesis. This process leads towards homozygosis and subsequent expression of the malignant trait (Wijnhoven et al. 2003, Preisler et al. 2000).
Our date demonstrate the recombinagenic effect of CEL in A. nidulans when a sensitive strain is used to study mitotic crossing-over. Further analyses, using mammalian cells, may be conduced for a better understanding of the carcinogenic potential of this solvent.
Thanks are due to Dr. Heloisa Helena Rodrigues de Andrade (Universidade Federal do Rio Grande do Sul) and Carmem Boto Querol (Universidade Estadual de Maringá) for supplying the cremophor EL; to Mrs. Sonia A. de Carvalho and Mrs. Luzia A. S. Regasse for technical assistance. Cleverson Busso is the holder of a PIBIC/CNPq fellowship.
BARRETT JC. 1993. Mechanisms of multistep carcinogenesis and carcinogen risk assessment. Environ Health Perspect 100: 9-12. [ Links ]
BÉGIN ML, ELLS G AND HORROBIN DF. 1988. Polyunsaturated fatty acid-induced cytotoxicity against tumor cells and its relationship to lipid peroxidation. J Natl Cancer Inst 80: 188-194. [ Links ]
BERGEN GG AND MORRIS NR. 1983. Kinetics of the nuclear division cycle of Aspergillus nidulans. J Bacteriol 156: 155-160. [ Links ]
BREIER A, DROBNÁ Z, DOCOLOMANSKÝ P AND BARANCÍK M. 2000. Cytotoxic activity of several unrelated drugs on L1210 mouse leukemic cell sublines with P-glycoprotein (PGP) mediated multidrug resistance (MDR) phenotype. A QSAR study. Neoplasma 47: 100-106. [ Links ]
BURTON AF. 1991. Oncolytic effects of fatty acids in mice and rats. Am J Clin Nutr 53: 1082-1086. [ Links ]
BUSSO C, CHIUCHETTA SJR, BAPTISTA F AND CASTRO-PRADO MAA. 2001. uvsH//uvsH diploid strain favors an efficient method to evaluate the recombinagenic effect of chemical and physical agents in Aspergillus nidulans (Ascomycetes). Acta Scientiarum 23: 603-607. [ Links ]
CHIUCHETTA SJR AND CASTRO-PRADO MAA. 2002a. Vincristine induces somatic segregation, via mitotic crossing-over, in diploid cells of Aspergillus nidulans. Biol Res 35: 31-38. [ Links ]
CHIUCHETTA SJR AND CASTRO-PRADO MAA. 2002b. Recombinagenic effect of Cryptolepine in uvsH+// uvsH+ and uvsH//uvsH diploid strains of Aspergillus nidulans. Folia Microbiol 47: 516-520. [ Links ]
DE LA TORRE RA, ESPINOSA-AGUIRRE JJ, CORTINAS DE NAVAS C, IZQUIERDO T AND MORON F. 1994. Genotoxic activity of mebendazole in Aspergillus nidulans. Mutat Res 305: 139-144. [ Links ]
FISHEL R AND KOLODNER RD. 1995. Identification of mismatch repair genes and their role in the development of cancer. Curr Opin Genet and Dev 5: 382-395. [ Links ]
FJÄLLSKOG M-L, FRII L AND BERGH J. 1993. Is Cremophor EL, the solvent for paclitaxel, cytotoxic? Lancet 342: 873. [ Links ]
FRANZONI MGM, CASTRO-PRADO MAA AND GEBARA JS. 1997. On the recombinagenic activity of Norfloxacin in a diploid strain of Aspergillus nidulans. Cytologia 62: 39-45. [ Links ]
FRICHE E, DEMANT EJF, SCHESTAD M AND NISSEN NI. 1993. Effect of anthracycline analogs of photolabelling of p-glycoprotein by 125I-iodomycin and 3H-azidopine: relation to lipophilicity and inhibition of daunorubicin transport in multidrug resistant cells. Br J Cancer 67: 226-231. [ Links ]
GELDERBLOM H, VERWEIJ J, NOOTER K AND SPARREBOOM A. 2001. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer 37: 1590-1598. [ Links ]
GOLDMAN GH, MCGUIRRE SL AND HARRIS SD. 2002. The DNA damage response in filamentous fungi. Fungal Genet Biol 35: 183-195. [ Links ]
HJERTVIK M, ERIXON K AND AHNSTRÖM G. 1998. Repair of DNA damage in mammalian cells after treatment with UV and dimethyl sulphate: discrimination between nucleotide and base excision repair by their temperature dependence. Mutat Res 407: 87-96. [ Links ]
HOFFMAN H. 1984. Polyoxythylenglycerol triricinoleat 35 DAC 1979. Pharm Zeit 129: 1730-1733. [ Links ]
IWANEJKO L, COTTON C, JONES G, TOMSETT B AND STRIKE P. 1996. nuvA, an Aspergillus nidulans gene involved in DNA repair and recombination, is a homologue of Saccharomyces cerevisiae RAD18 and Neurospora crassa uvs-2. Microbiology 142: 505-515. [ Links ]
KUNZ BA, BARCALY BJ AND HAYNES RH. 1981. Phenomenology and genetic control of mitotic recombination in yeast. Ann Rev of Genet 15: 57-80. [ Links ]
MAHER VM, BHATTACHARYYA NP, MAH MC, BOLDT J, YANG JL AND MCCORMICK JJ. 1993. Mutations induced by 1-nitrosopyrene and related compounds during DNA replication in human cells and induction of homologous recombination by these compounds. Res Rep Health Eff Inst 40: 41-51. [ Links ]
NOWELL P. 1976. The clonal evaluation of tumour cell populations. Science 194: 23-28. [ Links ]
NYGREN P, CSÓKA K, JONSSON B, FRIDBORG H, BERGH J, HAGBERG H, GLIMELIUS B, BRODIN O, THOLANDER B AND KREUGER A. 1995. The cytotoxic activity of Taxol in primary cultures of tumor cells from patients is partly mediated by Cremophor EL. Br J Cancer 71: 478-481. [ Links ]
OSMAN F, TOMSETT B AND STRIKE P. 1993. The isolation of mutagen-sensitive nuv mutants of Aspergillus nidulans and their effects on mitotic recombination. Genetics 134: 445-454. [ Links ]
PIRES LTA AND ZUCCHI TMAD. 1994. A new method to detect potential genotoxic agents using mitotic crossing over in diploid strains of Aspergillus nidulans. Bras J Gen 17: 371-376. [ Links ]
PRESILER V, CASPARY WJ, HOPPE F, HAGEN R AND STOPPER H. 2000. Aflatoxin B1-induced mitotic recombination in L5178Y mouse lymphoma cells. Mutagenesis 15: 91-97. [ Links ]
RAMEL C, CEDERBERG H, MAGNUSSON J, VOGEL J AND NATARAJAN AT. 1996. Somatic recombination, gene amplification and cancer. Mutat Res 353: 85-107. [ Links ]
ROPER JA. 1952. Production of heterozygous diploids in filamentous fungi. Experientia 8:14-15. [ Links ]
SENGSTAG C. 1994. The role of mitotic recombination in carcinogenesis. Crit Rev Toxicol 24: 323-353. [ Links ]
STERM C. 1936. Somatic crossing-over and segregation in Drosophila melanogaster. Genetics 21: 625-730. [ Links ]
VAN DE VATE C AND JANSEN GJO. 1978. Meiotic recombination in a duplication strains of Aspergillus nidulans. Genet Res 31: 29-52. [ Links ]
WIJNHOVEN SW, SONNEVELD E, KOOL HJ, VAN-TEIJLINGEN CM AND VRIELING H. 2003. Chemical carcinogens induce varying patterns of LOH in mouse T-lymphocytes. Carcinogenesis 24: 139-144. [ Links ]
WOODCOCK DM, JEFFERSON S, LINSENMEYER ME, CROWTHER PJ, CHOJNOWSKI GM, WILLIAMS B AND BERTONCELLO I. 1990. Reversal of the Multidrug Resistance Phenotype with Cremophor EL, a common vehicle for water-insoluble vitamins and drugs. Cancer Res 50: 4199-4203. [ Links ]
Manuscript received on June 13, 2003
Accepted for publication on October 8, 2003
Presented by Lucia Mendonça Previato