Article pubs.acs.org/est
Changes in the Expression of cyp35a family Genes in the Soil Nematode Caenorhabditis elegans under Controlled Exposure to Chlorpyrifos Using Passive Dosing Ji-Yeon Roh, Hwang Lee, and Jung-Hwan Kwon* Division of Environmental Science and Ecological Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 136-713, Republic of Korea S Supporting Information *
ABSTRACT: In order to use sensitive molecular-level biomarkers for the evaluation of environmental risks, it is necessary to establish a quantitative dose−response relationship. Passive dosing is regarded as a promising new technique for maintaining a constant exposure condition of hydrophobic chemicals in the assay medium. The main goals of the present study were (1) to quantitatively compare gene expression results obtained using the passive dosing method and the conventional spiking method and (2) to investigate changes in gene expression with respect to the free concentration and exposure duration using passive dosing. Chlorpyrifos (CP), which is oxidized by the cytochrome P450 monooxygenases, was selected as a model chemical, and the expression of cytochrome P450 subfamily protein 35A gene series (cyp-35a1− 5) was analyzed by quantitative real-time PCR on soil nematode Caenorhabditis elegans. Whereas the free concentration of CP rapidly decreased and the expression of cyp genes varied with the volume of exposure medium and the test duration when the spiking method was used, the free concentration in the assay medium was stable throughout the experiment when the passive dosing method was used. In addition, the level of gene expression increased with exposure time up to 8 h and with increasing CP concentration. The observed increased gene expression could be explained by increasing body residue concentration of CP with exposure time. In conclusion, quantitative dose−response relationships for gene expression biomarkers could be obtained for highly hydrophobic chemicals when the constant exposure condition is provided and the free concentration is used as the dose-metric.
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INTRODUCTION Environmental biomarkers, including genetic indicators, have been of interest for decades. Many researchers have investigated the use of biomarkers for the evaluation of ecotoxicity and the resulting environmental risks. Biomarkers reflect the initial responses of an organism exposed to a low concentration of a chemical stressor prior to the appearance of toxicological effects at the organism level (e.g., survival, behavior, and reproduction).1 Early biomarker studies focused mainly on the biochemistry based on enzyme activity, whereas, in recent years, the focus has shifted to genetics based on the transcriptome analysis, in which the specific amino acid sequence of protein production is included. With the accumulation of genetic information from many species in various media and the development of various analytical techniques such as real-time PCR, microarray, and gene sequencing, gene expression has emerged as a sensitive and specific end point for the evaluation of environmental risks.2,3 Gene expression in diverse species has been measured to monitor the environmental quality of soil, fresh water, salt water, and sediment.4−7 In gene expression studies, a reliable definition of the exposure conditions, such as the free concentration of a test chemical in the exposure medium and the test duration, is very © 2014 American Chemical Society
important, because the initial responses are affected by the exposure conditions at a low level of exposure. So far, many earlier studies denoted exposure concentration as the nominal concentration at initial exposure even when assessing highly hydrophobic organic chemicals such as chlorpyrifos, tributyltin, and octachlorostyrene.8−10 However, Escher and Hermens11 suggested that toxicological responses to chemicals correlate better with the internal exposure concentration, and many researchers agreed that nominal concentration is not a good dose-metric when evaluating the toxicity of hydrophobic organic chemicals.12−14 In many cases, the free concentration may be much lower than the nominal concentration because volatile compounds can be lost during the experiment and lipophilic chemicals can bind to the plastic surfaces of the test plate and in cellular matrices.15−18 To overcome the aforementioned problems, passive dosing has been proposed as a novel method for controlling the free concentration in the test medium.13,19−24 Unlike conventional spiking methods, in which chemicals are administered directly Received: Revised: Accepted: Published: 10475
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mL for tests in 6-well plates. To expose C. elegans to CP, worms were pooled in a micro tube (1 mL). After 1−2 min, during which the worms to sink to the bottom of the tube, 100 μL of worm condensate was transferred to the test medium. Worms were exposed for 2, 4, 8, 12, and 24 h at 20 °C, respectively, and the nominal concentration of CP was set at 0.3 mg L−1. Stock solution of CP dissolved in DMSO was spiked into the medium containing the worms. The amount of DMSO added was less than 0.1% of the total volume. Testing with the Passive Dosing Method. PDMS sheets were loaded with CP using a loading solution (methanol:water = 6:4 [v/v]) to achieve the desired concentration. Equilibrium distribution coefficient (K PDMS/loading solution = 10 2.51 ; KPDMS/medium = 104.47) between PDMS and the loading solution from preliminary measurements was used. The volume ratio of loading solution to PDMS was approximately 30. PDMS sheets loaded with CP were air-dried for 1 h to remove residual methanol in PDMS phase during the loading step. Then, a PDMS sheet loaded with CP was placed in each well of 24 or 6 well plates followed by the addition of 1.1 mL (for 24 well plates) or 10 mL (for 6 well plates) of K-media. The aqueous and PDMS phases were pre-equilibrated for 24 h before the beginning of the test. The worms were incubated in the medium at 20 °C for 8 h and sampled for PCR every 2 h. The intended exposure concentrations in the test medium using the passive dosing method were 0.03, 0.06, 0.15, and 0.30 mg L−1. The preliminary measured partition coefficient between PDMS and K-media was used to load PDMS with the desired concentrations of CP. Gene Expression. C. elegans was homogenized in a 7 mL glass Dounce tissue grinder (Wheaton, Millville, NJ), and total mRNA was extracted with a NucleoSpin RNA kit (MachereyNagel GmbH & Co., Düren, Germany) according to the standard protocol. Quantitative real-time PCR was performed in triplicate using an oligo(dT) primer (Bio-Rad Laboratories, Hercules, CA) and the iQ SYBR Green Supermix (Bio-Rad). Five selected genes were analyzed by PCR using an Eco RealTime PCR System (Illumina, San Diego, CA). The primers were designed according to sequences retrieved from the C. elegans database (www.wormbase.org; Table S1, Supporting Information (SI)). Efficiency and sensitivity tests were performed for each gene to optimize the quantitative realtime PCR conditions before the main experiment. Five replicates were conducted at each dose for quantitative realtime PCR analysis, and gene expression was normalized to the level of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, gpd-1. Statistical difference between the control and exposed worms was determined by a parametric t test. Chemical Analysis. Changes in the free concentration of CP in the medium were monitored during the solvent spiking and the passive dosing tests, with or without worms, as described below. The exposure medium (1 mL) was transferred to a glass vial containing a PDMS disk (6 mm in diameter, 1 mm thick), used as an extracting phase. The vial was shaken at 150 rpm at 25 °C for 24 h to extract CP. The PDMS disk was then placed in a vial containing 1 mL of ethyl acetate for 24 h. The ethyl acetate extract was analyzed with a Hewlett-Packard 5890 Series II gas chromatograph (GC) equipped with an electronic pressure control (EPC), a split/splitless capillary inlet, and an electron capture detector (ECD). One microliter of the extract was injected at the split ratio of 10:1. CP was separated on an HP-5 column (30 m × 0.25 mm i.d., 0.25 μm
once or multiple times, passive dosing relies on the controlled release of chemicals via passive diffusion from a medium with a high sorption capacity for the test chemical. Thus, the free concentration of a chemical in the medium in equilibrium with the dosing phase is maintained constant throughout the experiment. In addition, passive dosing does not require organic cosolvents, which are frequently used to administer highly hydrophobic organic chemicals to aqueous test solutions, even though they may have adverse effects on the test specimen.25 Therefore, passive dosing may enhance the applicability of gene expression as an early biomarker, especially when assaying highly hydrophobic chemicals. The primary goals of this study were to develop a passive dosing method for the evaluation of gene expression in response to a model hydrophobic organic chemical and to define a quantitative dose−response relationship under controlled exposure conditions. We measured xenobiotic metabolism-related gene expression in response to chlorpyrifos (CP) exposure by using a conventional spiking method and the passive dosing method in order to quantitatively compare the two exposure methods and to identify changes in gene expression when the free concentration of CP was maintained using the passing dosing method. Caenorhabditis elegans was chosen as a model species because previous studies have used genetic tools to investigate the organism’s toxicological responses.26−29 The expression of five cytochrome P450 subfamily genes of C. elegans was quantified using real-time PCR. Based on the linear increase in the expression of these genes, the lowest observed effect concentration (LOEC) of CP was determined. Polydimethylsiloxane (PDMS) was used as the dosing phase in the passive dosing method to maintain the desired aqueous concentration of CP. The free concentrations of CP in the test media were monitored for both dosing methods.
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MATERIALS AND METHODS Organism. A soil nematode, C. elegans, was used as the model species because it is the first multicellular organism whose genome has been completely sequenced, and it is widely used as a model species. The wildtype C. elegans Bristol strain N2 was maintained on nematode growth medium (NGM) at 20 °C and fed Escherichia coli strain OP50. Eggs were isolated from mature adults using a 5% hypochlorite solution, followed by a rinse with S-buffer (129 mL 0.05 M K2HPO4, 871 mL 0.05 M KH2PO4, 5.85 g NaCl), and the eggs allowed to hatch on agar plates with a food source. After 3.5 days, young adults were used for all experiments. K-media (0.032 M KCl and 0.051 M NaCl) was used for aqueous exposure. Materials and Chemicals. Chlorpyrifos (CP) was chosen as the model hydrophobic chemical. CP was purchased from Sigma-Aldrich (cat. 45359; St. Louis, MO), and medical grade PDMS sheets were purchased from Specialty Silicone Products Inc. (cat. SSP-M8232; Ballston Spa, NY). PDMS was cut into a rectangular sheet (11 mm × 47 mm for a 24-well plate and 11 mm × 109 mm for a 6-well plate) for passive dosing of CP. The custom-cut PDMS sheets were cleaned two times with nhexane and methanol for 2 h each and stored in methanol until use. For the spiking method, CP was dissolved in dimethyl sulfoxide (DMSO) to generate the stock solution, and the desired nominal concentration of CP was obtained by spiking the stock solution into K-media. Testing with the Conventional Spiking Method. The volume of K-media was 1.1 mL for tests in 24-well plates or 10 10476
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Figure 1. Changes in the free concentration of CP in the test medium using the spiking method (A) and the passive dosing method (B). The nominal concentration for the spiking method and the intended equilibrium concentration for the passive dosing method were 0.3 mg L−1. Error bars denote standard deviations of triplicate measurements; they are not shown if they are smaller than the size of symbols.
Figure 2. Quantitative analysis of the expression of cyp35a subfamily genes in wildtype C. elegans exposed to 0.3 mg L−1 of CP using two different methods. Worms were exposed to medium containing 0.3 mg L−1 of CP initially (A) and were transferred to a well containing a PDMS sheet loaded with CP to provide 0.3 mg L−1 of CP in the medium (B). Gene expression was analyzed in quantitative real-time PCR and was normalized to the gpd-1. Data are presented in arbitrary units relative to control (0 h = 1; n = 3; *p < 0.05). Error bars denote the standard error of the mean.
film thickness; Agilent J&C Scientific, Folsom, CA). The column oven temperature was held at 100 °C for 2 min, then increased to 290 °C at 25 °C min−1 and held for 2 min. The injector and the ECD temperature were 200 and 300 °C, respectively. Extraction recovery, evaluated at the three concentrations of CP spiked into the test medium (0.1, 0.3, and 0.5 mg L−1) and was 89.6 ± 4.4%. Average value of extraction recovery was applied to calculate the free concentration in the medium from the measured value.
sampling times regardless of the volume and the presence of C. elegans when passive dosing method was used (Figure 1B). Given that the water solubility and vapor pressure of CP at 20 °C are 0.4−2 g m−3 and 0.0022−0.0067 Pa, respectively,30 loss of CP via volatilization was negligible due to the low Henry’s law constant. Thus, the loss of CP from the medium in Figure 1A is likely due to sorption to the polystyrene plate and uptake by worms. Because polystyrene has a high sorption capacity for hydrophobic organic chemicals31 and because the volume of worms was much lower than that of the plate, sorption to the plate surface followed by slow diffusion into the plastic phase constituted the major mechanism for loss of CP when the spiking method was used. Furthermore, an increase in the plastic to water volume ratio explains the greater decrease in the free concentration of CP observed in tests with 24-well plates. On these plates, biological uptake accounts for the additional decrease in the free concentration in the presence of worms because a similar volume of worm condensate was added to each well regardless of the volume of K-media. When the passive dosing method was used, the free concentration of CP did not change significantly, regardless of the exposure conditions and despite sorption and biological uptake, because
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RESULTS AND DISCUSSION Measurement of the Free Concentration of CP in Two Different Dosing Methods. As shown in Figure 1A, static exposure with the spiking method resulted in a rapid decrease in the free concentration of CP, which may relate directly to toxicological responses. The free concentration dropped to about 20−75% of the initial value depending on the exposure. The decrease in the free concentration of CP was 13−23% greater in the 24-well plate assay than in the 6-well plate assay. In tests using 24-well plates, the presence of C. elegans led to an additional 7−10% decrease in the free concentration. In contrast, the free concentration of CP in the medium was maintained within ±10% of the intended concentration at all 10477
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phophorus pesticides via CYP to produce organophosphorus oxons that inhibits AChE activity in C. elegans is not fully understood. However, Menzel et al.37,38 reported that 80 different cytochrome P450 genes in C. elegans were identified and in particular, CYP35 forms, highly homologous to CYP2 forms in mammals, were induced by a various xenobiotics including PCB52, fluoranthene and lansoprazole. Roh and Choi29 suggested that C. elegans cyp-35a2 gene may be an important gene for exerting toxicity of fenitrothion, an orgnophosphorus pesticide that is structurally similar to CP. The increased expression of cyp-35a2 gene was accompanied by inhibition of AChE activity, developmental disturbance and increased mortality. However, development disorder by fenitrothion was significantly rescued, when the expression of cyp-35a2 was knocked down using RNA interference (RNAi). Furthermore, AChE acitivity was slightly less inhibited on cyp35a2 RNAi-treated worms compared to wildtype. These results suggest that cyp-35a2 may be involved in the oxidation of fenitrothion and toxicity in C. elegans.29 Among all cyp35a subfamily gene series (cyp-35a1−5), the expression of cyp-35a2 and cyp-35a3 genes is greater than other genes (cyp-35a1, cyp35a4, and cyp-35a5) by a factor of approximately 2 in this study. With the evidence of initial inhibition of AChE activity, this suggests that cyp-35a2 and cyp-35a3 genes are likely to be involved in metabolic toxicity of CP via CYP. Investigation of Changes in Gene Expression Using Passive Dosing. In test systems loaded with four different concentrations of CP, the free concentration remained at the intended concentration until the end of the experiment (Figure 3), as in the previous experiment (Figure 1B). The expression
PDMS contained large amounts of CP throughout the assay and losses to receiving phases were negligible. Identification of Toxic Effects at the Gene Expression Level. When the exposure concentration was maintained at a constant level using passive dosing, gene expression did not depend on the size of the exposure plate (6- or 24-well plate), whereas gene expression varied greatly according to the volume of the exposure medium when the same nominal concentration of CP was applied with the spiking method (Figure 2). After 0.3 mg L−1 of CP was spiked into 24-well plate, the expression of four genes (cyp-35a2, cyp-35a3, cyp-35a4, and cyp-35a5) increased to approximately twice that of the control (0 h) during the initial exposure phase (2 h), but decreased thereafter. In contrast, when the same nominal concentration of CP was used in 6-well plate, cyp-35a2 and cyp-35a3 expression steadily increased to approximately 1.5−2 times that of the control. Expression of cyp-35a4 and cyp-35a5 peaked at 1.5 times that of the control after 8 h and then decreased to no response level (Figure 2A). When C. elegans was exposed to CP using passive dosing, the expression of cyp-35a2, cyp-35a3, cyp35a4, and cyp-35a5 increased linearly over the test duration (Figure 2B). In particular, cyp-35a2 and cyp-35a3 expression increased to approximately 5 times that of the control after 8 h of exposure. The possibility of toxicity caused from PDMS without CP was confirmed as negligible effects on gene expression (Table S2, SI). Although CP is a lipophilic chemical with log KOW value of 5.0,32 it is rapidly transformed to an activated neurotoxic metabolite, chlorpyrifos-oxon (CPO), by cytochrome P450 enzymes.33 In other words, the parent form of CP is not likely to accumulate in the body. Thus, it is likely that the biologically available concentration of CP provided by external sources determines the metabolic activity in the body. Decrease in gene expression after up-regulation at initial time (2 h) is likely due to the rapid decrease in free concentration of CP surrounding C. elegans (Figure 1A), leading to a decrease in the concentration of CP in the body after the initial uptake; in the absence of CP metabolism, cytochrome P450 is not needed. Gene expression increased over a longer time in 6-well plate tests, in which the free concentration declined at a slower rate than in 24-well plate tests. The fact that the expression of cyp genes were increased linearly over the exposure duration, when the free concentration was maintained at a constant level using passive dosing, also supports this claim. Gene expression could not be analyzed after 10 h of exposure to 0.3 mg L−1 of CP using the passive dosing method because visible deformations of C. elegans were apparent. As shown in Figure S1 (SI), worms were coiled in the middle of body after 12 h of exposure, and they died after 24 h with body rupture. The reference value of 0.3 mg L−1 of CP corresponds to the LC10 value calculated using the nominal concentration in a previous study at the same exposure duration,34 at which no significant changes in the survival of C. elegans were observed. By inhibiting acetylcholinesterase (AChE) activity, CPO causes the neurotransmitter acetylcholine to accumulate in neural junctions.35,36 Such cholinergic toxicity could cause loss of body functions and abnormal behaviors and eventually result in fatality. Although level of CPO was not measured in this study, the AChE activity was verified (Figure S2, SI). The AChE activity, which is involved in the toxic mechanism of organophosphorus pesticide, was inhibited about 70−80% compared to that of control at 4 h after the exposure using passive dosing (Figure S2, SI). Metabolism of organo-
Figure 3. Changes in the free concentration of CP in the test medium using the passive dosing method. The intended equilibrium concentrations were 0.03, 0.06, 0.15, and 0.3 mg L−1.
of cyp-35a2 and cyp-35a3 genes increased from the onset of exposure (Figure 4). Again, cyp-35a1 did not show any response within the range of CP concentrations investigated, whereas cyp-35a4 and cyp-35a5 expression increased with exposure time, reaching levels approximately 2-fold higher than in control (Figure S3, SI). Thus, cyp-35a2 and cyp-35a3 were chosen as markers because they were most sensitive to CP and their responses were quantifiable at different concentrations and exposure times. After 8 h of exposure to 0.06, 0.15, and 0.3 mg L−1 CP, cyp-35a2 expression was 2.2, 3.3, and 4.7 times that of the control, respectively. Similarly, cyp-35a3 expression increased linearly over time, but to a lesser extent (1.9-, 2.9-, 10478
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Figure 4. Quantitative analysis of cyp-35a2 and cyp-35a3 expression in wildtype C. elegans exposed to each intended equilibrium concentration of CP (0.3, 0.15, 0.06, and 0.03 mg L−1) using the passive dosing method. Worms were extracted at 0, 2, 4, 6, and 8 h. Gene expression was analyzed in quantitative real-time PCR and was normalized to the gpd-1. Data are presented in arbitrary units relative to control (0 h = 1; n = 5; *p < 0.05). Error bars denote the standard error of the mean.
Table 1. Comparison of Studies of Chlorpyrifos Toxicity in Caenorhabditis elegans exposure conditionb
cosolvent
measured/nominal
K-media K-media K-media K-media K-media NGM K-media K-media K-media
none none DMSO acetone DMSO acetone acetone none acetone
N N N N N N N M N
toxic end pointa movement movement behavior mortality feeding gene expression (microarray) cyp-35a2 gene expression cyp35a subfamily gene expression development
24h-LOEL 72h-LOEL 4h-EC50 24h-LC50 24h-EC50 72-LOEL 24h-LOEL 8h-LOEC 96h-LOEL
value
reference
3.0 mg L−1 3.0 mg L−1 1.4 mg L−1 1 mg L−1 0.35−0.77 mg L−1 0.5 mg L−1 0.1 mg L−1 0.06 mg L−1 0.01 mg L−1
39 39 42 34 43 44 34 this study 34
a
EC50: median effective concentration; LC50: median of lethal concentration; LOEL: lowest observed effect level; NOEL: no observed effect level; LOEC: lowest observed effect concentration. bNGM = nematode growth medium.
and 3.7-fold change at 0.06, 0.15, and 0.3 mg L−1 CP, respectively). As shown in Figure 4, cyp-35a2 and cyp-35a3 expression increased linearly with increasing exposure duration and free CP concentration. However, the response to 0.03 mg L−1 was minimal. The increases in gene expression could be explained by the bioconcentration of CP in the body. Although cytochrome P450 enzymes metabolize and eliminate CP, the CP uptake rate may compete with the CP elimination rate when a constant free concentration of CP is maintained by passive dosing because residual CP would increase continually in the body up to a certain exposure time. Consequently, C.
elegans must continue to produce xenobiotic enzymes leading to increased expression of xenobiotic-related mRNA over time. These results are different from those obtained by the spiking method, in which cyp-35a gene expression decreased after an initial increase (Figure 2A). This suggests that more reliable and toxicokinetically meaningful gene expression data can be obtained for hydrophobic organic chemicals that metabolized in the body. Comparison of the Results with Literature. When gene expression levels were measured at different exposure concentrations and durations, the LOEC of CP was 0.06 mg 10479
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L−1. Table 1 compares toxicity values reported for chlorpyrifos in C. elegans using various end points. Most of the earlier results were obtained using higher-level parameters of biological organization (i.e., mortality, growth, movement, and reproduction), and the toxicity values were greater than the LOEC in this study. For example, Ruan et al.39 used movement as a toxicological end point and proposed a 24 h lowest observable effect level (LOEL) of 3 mg L−1, which even exceeds the water solubility of chlorpyrifos at 20 °C (0.73 mg L−1).32 The toxicity values calculated by using gene expression or protein levels as the toxicological end point were below 0.1 mg L−1, close to the LOEC in this study.34 Although the concentration reported by Roh and Choi34 using development as the toxicological end point is slightly lower than the LOEC in this study, long-term exposure (>96 h) was required, and the toxicological value was obtained over multiple generations. Thus, the method proposed in this study presents a promising way to use gene expression as a sensitive biomarker for hydrophobic chemicals without use of a cosolvent or long-term exposure. Although the effect level in C. elegans in this study was lower than most of literature values by approximately an order of magnitude, C. elegans is relatively insensitive to specific toxicity of CP compared with values reported in literature using other organisms such as Daphnia magna.40,41 However, the LOEC value obtained from gene expression level response in this study would be still useful in the assessment of ecotoxicological risks in the terrestrial environment. Environmental Relevance of the Findings. To use sensitive and specialized biomarkers as end points for environmental risk assessment, it is necessary to clearly define and quantify the exposure conditions. The free concentration of chemical in the test medium, which directly relates to the toxicity, could be much lower than expected, depending on the chemical properties and experimental conditions, when the chemical is introduced by solvent spiking. This makes it difficult to use many earlier studies for risk assessment purposes especially for hydrophobic organic chemicals. However, as shown in this study, a quantitative dose−response relationship can be derived for well-known cyp genes that respond to xenobiotics at exposure concentrations lower than those reported in earlier studies if a constant exposure concentration is maintained by using the passive dosing method. Although further studies are needed, the results in this study support the use of gene expression as a promising early biomarker for chemical risk assessment when regulation at the genetic level triggers toxic responses and the adverse outcome pathways are simple.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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REFERENCES
We thank Dr. Jinhee Choi (School of Environmental Engineering, University of Seoul.) for supplying C. elegans. This research was supported by the National Research Foundation of Korea (NRF) (grant number 2012R1A1B4000841 and 2013R1A1A2060473).
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ASSOCIATED CONTENT
* Supporting Information S
Genes and primers used for qPCR are listed in Table S1. Quantitative gene expression results for PDMS control test are shown in Table S2. Microscopic images of C. elegans are shown in Figure S1. Acetylcholinesterase activity is shown in Figure S2. Quantitative gene expression results for all genes tested are shown in Figure S3. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*=Phone: +82 2 3290 3041; fax: +82 2 953 0737; e-mail:
[email protected]. 10480
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dx.doi.org/10.1021/es5027773 | Environ. Sci. Technol. 2014, 48, 10475−10481
