Cremophor EL stimulates mitotic recombination in uvsH / / uvsH diploid strain of Aspergillus nidulans

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 G2 period, 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).


INTRODUCTION
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 Pglycoprotein, 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 as-
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 andMorris 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.

Strains
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).

Culture Media
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 37˚C.

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 37˚C 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.

Cytological Analysis
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 37˚C, 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 37˚C.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 trans-ferred to 25 defined positions in CM plates (master plates).After 48 hours of incubation at 37˚C, colonies were transferred to selective media and the phenotypical analysis of the haploid segregants was carried out.
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 pyridoxinesupplemented 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.

TABLE I Genotype and origin of strains.
). FGSC -Fungal Genetic Stock Center, University of Kansas Medical Center, Kansas City, USA.

TABLE II Mitotic segregation of paba gene among haploid segregants derived from paba+//paba heterozygous diploid in the presence and in the absence of crossing-over.
(*) Homozygous diploid (paba // paba) will not be chosen in MM + pyridoxine plates

TABLE III Time of conidiophore vesicle, metulae and phialides appearance and conidia production of B211 strain in the presence of cremophor EL 0.05 and 0.075% v/v.
*Development without cremophor EL.