Fig 1.
Transcriptional responses for mRNA encoding CYP following in vitro exposure of Opisthorchis felineus to xenobiotics.
Analysis by real-time PCR with normalization based on the expression stability value (M-value); normalization was undertaken using Ub and MrpL16 genes as endogenous internal controls (M < 1.2). Triplicate real-time PCRs were run for each sample. A. An adult worm; B. Newly excysted metacercariae (NEM). C. CYP mRNA level after in vitro treatment of O. felineus for 20 h with hemoglobin (DDPCR). CYP gene expression levels were quantified and values were simultaneously normalized to reference gene MrpL16 expression using QuantaLife (Bio-Rad, USA). The data are shown as the normalized ratio of CYP to MrpL16 ± S.D. Each run was made in duplex. Results of three independent experiments are presented.
Fig 2.
Representative chromatogram of chlorzoxazone metabolism in Opisthorchis felineus.
(a scanned copy of the original print version). A. extract of O. felineus incubation media without chlorzoxazone; B. extract of O. felineus incubation media containing 10 mM chlorzoxazone; C. extract of O. felineus incubation media containing 10 mM chlorzoxazone and 40 μМ ketoconazole; A peak corresponding to retention time of 6OH-CLZ in the 2c chromatogram is indicated with an arrow. D. extract c spiked with a standard of 6- hydroxychlorzoxazone; E structures of 6-OH-hydroxychlorzoxazone (1), chlorzoxazone (2), benzoxazole (is); F. Analysis of relative 6OH-CLZ amount. The area of the 6OH-CLZ peak in each chromatogram was measured. Data were expressed as a fold difference compared to the control. Data are presented as means ± S.D. Results are averaged from three independent experiments.
Fig 3.
Fluorescence micrographs of Opisthrochis felineus after exposure in vitro for 20 h to several substances. A. A control fluke (multiple fluorescence filters: FITC, rhodamine, DAPI). B. Adult worms were treated with methoxyresorufin. C. Adult worms were treated with benzoxyresorufin. D. Adult worms were treated with pentoxyresorufin (PR). The resorufin (R) that was formed in fluke tissues is indicated with an arrow (FITC, rhodamine fluorescence filters). E. Adult worms were treated with penzoxyresorufin (rhodamine). F. Excretory granules of the resorufin formed in O. felineus treated with pentoxyresorufin. Monochrome images were acquired using a rhodamine filter (5F is a part of image 5E). G. A worm was treated with PR and ketoconazole (rhodamine filter).
Fig 4.
Suppression of expression of CYP in adult Opisthorchis felineus by RNA interference (RNAi).
A. Transcript levels were determined using EVA-green real-time RT-PCR. The MrpL16 gene was used for normalization. The control group (no dsRNA treatment) was compared to the negative control group transformed with LUC dsRNA. Three biological samples with technical duplicates were used for analysis. The data are presented as means ± SD. B. Primer positions for analysis of CYP gene expression. C. Control adult O. felineus, eight days after electroporation. EC: excretory channel, EB: excretory bladder. D. Worms eight days after of the knockdown of CYP mRNA. E. Percentage of worms with phenotypic changes in excretory system after three days of RNA interference (data of three independent experiments). Wild type–untreated worms; mock–worms subjected to electroporation without dsRNA; LUC–worms that received LUC dsRNA (non specific control); CYP–worms that received CYP dsRNA; keto—worms were treated with ketoconazole for three days. F, G. Worms five days after the knockdown of CYP (F) and LUC (G) mRNA were treated for 20 h with pentoxyresorufin, and images acquired using multiple fluorescence filters using optical sectioning by means of the AxioImager fluorescence microscope (Zeiss). The resorufin (R) forming in the excretory bladder is indicated with an arrow. Representative images are shown. H. Adult O. felineus after three days of treatment with 40 μM ketoconazole.
Fig 5.
Kaplan-Meier survival curves and pentoxyresorufin metabolizing activity in worm tissues after RNA interference.
A. A set of Kaplan-Meier survival curves (out of three independent experiments) is shown. Statistical difference in survival log-rank (Mantel-Haenszel) test between each pair of samples was calculated. The survival curves show significance when either the wild type (p<0.0001) or LUC (p<0.001) or mock control (p = 0.002) is compared to CYP group and no difference is observed when LUC group and mock control are considered (p = 0.7)('survival'(v.2.38) R package). B. Analysis of pentoxyresorufin (PR) metabolizing activity. Worms five days after the knockdown were treated for 20 h with pentoxyresorufin, and images were acquired using multiple fluorescence filters using optical sectioning by means of the AxioImager fluorescence microscope (Zeiss). The total size of the resorufin particles in each worm was measured. Data were expressed as a fold difference compared to the wild-type (not exposed to dsRNA) control. Data are presented as means ± S.D. Results are averaged from three independent experiments. Significance at p < 0.005, ***; p < 0.01, ** (F-test).