1. IDENTIFICATION AND CHARACTERIZATION OF PHASE I
DETOXIFICATION ENZYMES IN ISOLATES OF FASCIOLA HEPATICA SUSCEPTIBLE AND RESISTANT TO TRICLABENDAZOLE
Pamela Lamenza1,2, Pedro Ortiz Oblitas4, Carolina Ceriani1,3, Hugo Solana1,2
Total RNA was extracted using Trizol ® (Invitrogen), from strains of F. hepatica TCBZ sensitive and resistant. This RNA was used for RT-PCR analysis.
The quality and quantity of RNA was assessed by agarose gel 1% visualized with SYBR Safe ® and spectrophotometrically through the A260nm/A280nm ratio.
We used specific oligonucleotides designed to conserved regions of the gene fmo that amplify a fragment of approximately 720 bp: 5'-fmo GAGAAATCTTGTGATATTGGTGGATTGTGG-Fv-3 '; fmo-Rv 5'-CCGTATCAGTTTCGATTGAATGGTCC-3'.
To compare the different conditions of RT-PCR was used cur gene (actin) as an internal control. Thermodynamic analysis of oligonucleotides was performed with the program OligoTech aided by DNAstar software.
1 Centro de Investigación Veterinaria de Tandil (CIVETAN) CONICET, 2 Laboratorio de Biología Celular y Molecular. FCV-UNCPBA, Tandil, Argentina
3 Laboratorio de Virología, FCV-UNCPBA, Tandil, Argentina , 4 Facultad de Ciencias Veterinarias. Universidad Nacional de Cajamarca, Cajamarca, Perú.
INTRODUCTION
The helminthes possess biochemical mechanisms for detoxification. Overall, parasites may evade anthelmintic effects by: i) mutation of target receptors, ii) overexpression of efflux transport pumps, and/or iii) overexpression of metabolic enzymatic systems. Fasciolosis is a zoonotic disease caused by Fasciola hepatica. Its control is mainly based on the use of triclabendazole (TCBZ), a halogenated benzimidazole thiol derivative which shows efficacy against juvenile and adult stages. The intensive use of TCBZ has resulted in the development of resistant liver flukes. In nematodes the resistance to benzimidazoles is caused by genetic changes in the β-tubulins. In F. hepatica the resistance to TCBZ has not been demonstrated by genetic changes and it is thought to be metabolically mediated. In general, all organisms have xenobiotic metabolizing enzymes, which can deactivate toxic functions. Within this group the most important Phase I detoxification enzymes are Cytochrome P450 (Cyt P450) and Flavin-monooxygenases (FMOs). In F. hepatica, there is a lack of knowledge about the participation of these enzymes in the mechanisms of resistance to TCBZ. This work was aimed to assess, by RT-PCR, the characterization and expression of FMO and P450 in F. hepatica susceptible and resistant toTCBZ.
MATERIAL & METHODS
Susceptible adult flukes (Cullompton Strain) and resistant (Sligo strain) to TCBZ were obtained from the bile duct of sheep inoculated orally with 200 metacercariae. Infection was confirmed 16 weeks after inoculation, assessing the presence of eggs by fecal analysis and determination of liver disorders by quantitation of GLDH activity (glutamate dehydrogenase) and gamma GT (glutamyl transferase) in serum.
Total RNA was extracted using Trizol ® (Invitrogen), from adult F. hepatica susceptible and resistant to TCBZ. This RNA was used for RT-PCR analysis. The quality and quantity of RNA was assessed by 1% agarose gel with SYBR Safe ® and spectrophotometrically through the A260nm/A280nm ratio.
The cDNA fragments generated by RT-PCR reactions were separated by electrophoresis on agarose gels and purified using the corresponding kit. Purified cDNA fragments were ligated to a commercial vector TOPO TA cloning kit (Invitrogen). Competent cells of E. coli DH5alpha was transformed with the recombinant plasmid, using TOP10 cells by heat treatment in the presence of CaCl2. The cells were grown in SOB medium for 1 hour at 37 ° C and seeded in Petri dishes containing LB supplemented with ampicillin. Positive clones were subjected to plasmidic extraction and plasmids were sent to external services sequenced by ABI PRISM sequencer using oligonucleotides that hybridize in the vector.
RESULTS
For the identification and characterization of fmo and Cyt P450 the RT-PCR was performed from specific oligonucleotides designed for that purpose. Bands of 720bp (gen fmo) and 930bp (gen Cyt P450) were obtained in the susceptible(Cullompton) and the resistant strain (Sligo). In turn, when analyzed comparatively both strains, in the case of fmo significant difference between both strains (S and R) was detected. In the resistant strain, the mRNA expression was 0.5 times higher than in the susceptible strain (quantified by ImageJ ®). No significant differences were detected in the expression of the RNAm of the Cyt P450 enzyme (data not show).
PCR reactions program
To obtained cDNA we used specific oligonucleotides, designed to conserved regions of fmo and cytP450, to amplify a fragment of 742 bp (gen fmo) and 939 bp (gen cyt P450) The specific oligonucleotides were designed from sequences of related organisms. Both RNAm for FMO and Cyt P450 were identified in F. hepatica genome. The expression of both enzymes was evaluated comparatively using the ImageJ®.
actin Kb
fmo
0,7 S R
cDNA corresponding to the susceptible (S) and resistant (R) isolate of adult F. hepatica.
fmo: Flavin Mono-oxygenase (FMO)
actin: Internal control.
mRNA expression levels of fmo:
The relative values ​​of mRNAs were quantified with the software ImageJ®, from cDNA bands of fmo relativized to cDNA bands of actin. (n = 3)
Levels of transcripts relativized to the control:
susceptible
resistant
DENATURATION ºC min ºC 5 min Start cycle
DENATURATION °C sec ºC 30 sec.
ANNEALING ºC sec ºC sec.
AMPLIFICATION ºC min ºC 1 min. 20 sec.
AMPLIFICATION ºC min ºC min End cycle
30 cycles
FMO
Cyt P450
Gen actin was used to compare the different conditions of RT-PCR, as an internal control.
Thermodynamic analysis of oligonucleotides was performed with the program OligoTech aided by DNAstar software.
MCS
pCytP kb
CytP450
pCR®-TOPO 3.9kb
RT-PCR
3900pb
930pb
1) RT-PCR
2) pCytP450 + EcoRI PM 1 2
DISCUSION AND CONCLUSION
Our previous results showed that in the case of F. hepatica TCBZ resistant (strain Sligo), the expression of anthelmintic resistance phenomenon is caused by an increased expression of FMO detoxificative pathway, this expression would be due to point mutations or a respective FMO increased expression of the synthesis of this enzyme.
These results confirmed that some members of the Phase I of detoxification not only are involved in the detoxification mechanisms, but also participate actively in the development of resistance to TCBZ in F. hepatica.
Generation of recombinant plasmid of Cyt450 from cDNA amplified by specific oligonucleotides
CIVETAN