유전자 발현과 환경 독성 번역

유전자 발현과 환경 독성 번역(한국어 원본)

ABSTRACT.
유전자 발현과 같이 민감한 분자수준의 생체지표를 환경위해성평가에 사용하기 위해서는 정량적 용량-반응관계의 성립이 요구된다. Recently, passive dosing is thought to be a promising new technique that provides a constant exposure condition of hydrophobic contaminants in the assay medium. The main goals of the present study were 1) to provide a quantitative comparison of the gene expression results obtained from the passive dosing method and the conventional spiking method and 2) to investigate the changes in gene expression with respect to the freely dissolved concentration and the exposure duration using passive dosing. CYP450 monooxygenases에 의해 산화되는 것으로 잘 알려진 CP를 target chemical로 선정하고, 이를 토양선충Caenorhabditis elegans의 cytochrome p450 family protein 35A gene series (cyp35a1-5)을 분석하였다. Spiking method를 이용한 실험 결과에서, CP의 자유농도는 급격히 감소되었으며, cyp family gene들은 총 노출 미디어 양에 따라, 노출 시간에 따라 expression pattern이 매우 다양하게 변화하였다. 그에 반해 passive dosing method를 적용한 경우에는 노출 실험이 종료되는 시점까지 자유농도가 안정적으로 C. elegans에게 제공되었으며, 시간에 따라 gene expression이 선형적으로 증가하였다. 또한, 일정하게 제공되어지는 CP 농도를 감소시키자, 시간에 따라 증가하는 경향의 기울기가 유의적으로 감소하였다. The increased gene expression with increasing body residue concentration of CP in accordance with the accumulation of CP in C. elegans may explain the observed effects at low concentration. In conclusion, quantitative dose-response relationships for gene expression biomarkers could be derived when constant exposure condition is provided and free concentration is used as the dose-metric.

Introduction
Gene expression은 환경 생태 위해성 평가 및 환경 독성 모니터링을 위한 매우 민감하고 중요한 종말점으로서의 잠재적인 가능성이 있다고 주장되어져 왔다. 그 이유는 gene expression이 생물반응 단계의 상위레벨 인 개체군 수준의 독성 영향(survivial, behavior, reproduction)이 나타나기 이전에 환경의 저농도 노출 환경에서 초기 독성 반응을 반영하기 때문이다(ref.). 이러한 높은 민감도로 인해 다른 어떠한 종말점보다도 노출 농도 또는 노출 기간과의 용량-반응관계가 명확해야하며, 이를 위한 정량적인 방법이 제공되어져야한다. 현재는 노출 조건이 잘 정의되어 있다 하더래도, 대부분의 유전자 발현을 이용하는 연구의 노출 농도가 초기 노출 농도의 nominal concentration으로 표기되고 있다. 그러나, 초기 노출되어진 nominal aqueous concentration은 노출 plate의 벽에 흡착되거나, 미디어내 components와 binding되거나, 증발하여 대량으로 감소된다. 실제적으로는 오직 freely dissolved concentration만이 target organism의 반응에 관여하게 된다. 이에 gene expression을 이용하여 독성 연구를 진행함에 있어 명확하게 정의되어진 freely dissolved concentration을 안정적으로 제공하여 줄 수 있는 노출 환경을 구성하는 것이 매우 중요하다 하겠다.
최근에는 수계 노출 환경에서 freely dissolved concentration을 제어하는 새로운 독성 노출 방법으로 passive dosing/sampling method가 제안되어지고 있다. 이 방법은 기존에 화학물질을 미디어에 일회 또는 여러 번에 걸쳐 직접적으로 투여하던 방법과는 달리 화학물질이 저장되어 있는 고분자로부터 passive diffusion에 의해 노출환경으로release된다. 고농도 dosing phase로부터 지속적으로 화학물질이 제공되어지는 이 원리는 다양한 경로로의 화학물질의 손실을 극복하고 실험 종료 시점까지 일정한 자유농도를 생물종에게 제공하여 줄 수 있도록 하였다. 특히, hydrophobic한 chemical의 경우, 수용해도 이상의 고농도 spiking stock을 조제하기위해 사용되어지 solvent가 passive dosing method에서는 필요하지 않으므로, 실험 생물종은 solvent에 coexpose되지 않을 수 있다.
본 연구에서 우리는 1)cytochrome p450 gene의 expression 을 이용하여 기존에 사용하던 spiking method 와 passive dosing method로 각각 제공한 노출환경에서 chlorpyrifos의 독성영향이 어떻게 다르게 나타나는 지를 비교함으로써 두 노출 방법에 대한 quantitative comparision을 제공하고, 2) passive dosing을 이용하여 안정적인 노출 환경을 제공할 때 자유농도와 노출 기간의 변화에 따른 gene expression의 변화를 조사하려고 한다. 이를 위해 동일한 농도로 의도되어진 노출환경에서 시간이 흐름에 따른 미디어 내 CP의 자유농도를 모니터링하고, 해당 노출 시간별로gene expression의 변화를 각각 rt-PCR로 분석하였고, 다양한 농도로 loading되어진 PDMS를 dosing phase로 제공하여 gene expression과 자유농도와의 용량-반응관계를 정량화하였다.

MATERIALS and METHODS
C. elegans는 NGM media에서 OP50을 먹이로 하여 20도에서 maintain하였다. 모든 실험에는 age synchronized culture하여 얻은 young adults(3days old)를 사용하였으며, k-media를 이용하여 수계 노출을 하였다.

CP는 sigma-aldrich에서 구매하였고, PDMS는 ~~에서 주문하였다. 이는 높이 11mm, 길이는 XX와 109mm로 24well과 6well의 내경에 두를수 있도록 두가지 size로 각각 cut하였고, 이 PDMS의 mass는 각각 0.5611, 1.3179g (n=30, standard deviation [SD] =0.0138g, 0.0243g)으로 측정되었다. 실험시 spiking을 하기 위해서는 DMSO를 solvent로 사용하여 stock을 제조하였으며, media에 투입된 solvent의 양은 전체 노출 media에 0.1%가 되도록 사용하였다.

Passive dosing method을 이용한 독성 실험을 위해서는 CP를 loading한 PDMS를 노출 plate에 장착하여 미디어 내로 의도한 CP가 release되도록 하여야한다. 이러한 과정은 노출 system이 평형상태일때, PDMS와 미디어 사이의 mass balance 식으로 아래와 같이 표현할 수 있다.
여기서, medium 에 release되는 CP의 양은 PDMS와 미디어간의 평형분배현상에 의해 결정되어지므로, 이를 고려한 식 3으로 표현 할 수 있다.
수식에서 알 수 있듯이, 은 노출 직전 초기의 CPDMS의 농도에의해 좌우된다. 원하는 CP 농도를 PDMS에 loading하는 것은 CP의 PDMS와 loading solution(methanol: water, 6:4 v/v)간의 분배계수에 의해 결정된다. PDMS와 loading solution간의 분배평형 상태는 150rpm으로 20도에서 24시간 shaking incubation시키는 것으로 충분히 도달하는 것을 확인하였다. That was determined by measuring the concentration in PDMS (CPDMS,0, mg/L) and the loading solution (Cloading solution, mg/L) independently.
최종적으로, 실험 미디어 내에 원하는 CP의 농도를 노출하기 위한 은 서로다른 두 평행 분배계수와 주어진 조건으로 식 5와 같이 나타 낼 수 있다.
여기서, KPDMSsm(6:4)는 PDMS와 sm(6:4)의 분배계수, KPDMSm는 PDMS와 미디어 사이의 분배계수를 의미한다. Csm은 sm(6:4)에 투여된 CP의 농도(mg/L), Vm과 VPDMS은 미디어와 PDMS의 부피(ml)이다.

미디어 총 노출 볼륨은 24well plate에 1ml, 6well plate에 10ml로 차등을 두어 실시하였으며, 노출 worm은E-tube에 pooling후 1~2분 간격을 두어 worm이 가라앉은 뒤 100ul 볼륨을 기준으로 총 노출 organism양을 결정하였다. Chlorpyrifos노출 농도는 0.3mg/L으로 하였으며(CP의 20도 기준으로 water solubility는 ~~이다.), spiking method를 사용한 실험은 worm이 들어있는 media에 spiking하여 바로 실험을 진행하였고, Passive dosing방법을 사용한 실험은 loading된 PDMS를 plate에 장착하여 24시간동안 분배평형이 이루어지도록 둔뒤, worm을 노출시켰다. 노출실험은 20도 에서 24시간 incubation하며 실시하였다. 이후 passive dosing method의 노출농도는 0.3mg/L로부터 1/2, 1/5, 1/10수준의 농도로 변화하며 실시하였으며, 노출시간은 실험 시작 후 2시간 간격으로 8시간까지 실시하였다.

노출 media내의 CP농도는 PDMS로 재 추출하여 분석하였다. 1ml의 노출 media를 vial에 옮긴뒤 직경 ~mm에 해당하는 disc PDMS를 넣어 24시간동안 150rpm으로 25도에서 shaking하며 PDMS내로 CP를 추출한뒤 disc PDMS를 동량의 Ethyl acetate로 치환하여 동일한 조건에서 24시간을 두어 역 추출을 실시하였다. 이를 GC로 분석하였으며, 추출 오차를 위한 recovery test는 3개의 농도에서 10회씩 실시하여 검증하였다.

유전자 발현과 환경 독성 번역(영어 번역본)

ABSTRACT

In order to apply sensitive molecular-level biomarkers to the evaluation of environmental risks, it is necessary to establish a quantitative relationship between dose and response. Recently, passive dosing is thought to be a promising new technique that provides a constant exposure condition of hydrophobic contaminants in the assay medium. The main goals of the present study were 1) to provide a quantitative comparison of the gene expression results obtained from the passive dosing method and the conventional spiking method and 2) to investigate the changes in gene expression with respect to the freely dissolved concentration and the exposure duration using passive dosing. Chlorpyrifos (CP), which is known to be oxidized by the CYP450 monooxygenases, was selected as the target chemical, and Caenorhabditis elegans cytochrome p450 family protein 35A gene series (cyp35a1-5) was analyzed. Freely dissolved concentration of CP rapidly decreased when spiking method was used, and the expression patterns of cyp family genes varied greatly with total volume of exposure media and exposure time. In contrast, freely dissolved concentration of CP provided to C. elegans was stable until the end of exposure experiment when passive dosing method was used, and gene expression linearly increased with time. When the constantly maintained concentration of CP was lowered, slope of the linearly increasing trend was significantly reduced. The increased gene expression with increasing body residue concentration of CP in accordance with the accumulation of CP in C. elegans may explain the observed effects at low concentration. In conclusion, quantitative dose-response relationships for gene expression biomarkers could be derived when constant exposure condition is provided and freely dissolved concentration is used as the dose-metric.

INTRODUCTION

It has been asserted that gene expression has the potential to be a very sensitive and important end point for evaluation of environmental risks and monitoring of environmental toxicity. The basis for this argument is that gene expression reflects the initial toxicity response under low-concentration exposure prior to the development of toxicological effect at the level of an individual organism (survival, behavior, reproduction, etc.), which is a higher level of biological response [REF]. Due to such high sensitivity, dose-response relationship between exposure concentration and duration must be clearly delineated in a quantifiable manner for gene expression to be used as an end point for this purpose. Currently, most gene expression studies, even those with well-defined exposure conditions, denote exposure concentration as the nominal concentration of the initial exposure concentration. However, the initial nominal aqueous concentration rapidly decreases due to adsorption to the exposure plate wall, binding to components in the media, or evaporation. In reality, it is the freely dissolved concentration that is directly related to the response of the target organism. From these considerations, it is apparent that an exposure environment that can provide sustained level of clearly defined freely dissolved toxin concentration is very important for toxicological research.
Recently, passing dosing and sampling method have been proposed as a novel toxin exposure method for controlling freely dissolved concentration in aqueous exposure environment. Unlike previous methods in which the chemical is directly released at once or over multiple runs, this method relies on controlled release of chemicals from polymers via passive diffusion. By allowing sustained supply of the chemical from the start of high-concentration dosing phase, loss of the chemical through various routes is minimized and freely dissolved concentration is maintained at a constant level until the end of the experiment. Especially for hydrophobic chemicals, high-concentration spiking stock with concentration above the chemical’s water solubility requires use of organic co-solvent that may have adverse effect on the test specimen, whereas passive dosing method does not require such organic solvent.
In our study, we used 1) cytochrome p450 gene expression to compare the toxicological effects of chlorpyrifos using the existing spiking method versus using the passive dosing method, thereby quantitatively comparing the two exposure methods. Also, we used 2) passing dosing to provide stable exposure environment and identify changes in gene expression when freely dissolved concentration and exposure duration are changed. Toward this goal, concentration of free CP in the media in which the concentration was set to be constant was monitored over time, and for each exposure time points, changes in gene expression were analyzed using rt-PCR. Also, PDMS with various concentrations were provided as the dosing phase to quantify the dose-response relationship between gene expression and freely dissolved concentration.

MATERIALS and METHODS

C. elegans was cultured in NGM media at 20 °C while feeding on OP50. C. elegans young adults (3 days old) obtained from age-synchronized culture were used for all experiments, and k-media was used for aqueous exposure.

CP was purchased from Sigma-Aldrich and PDMS was purchased from XXX. It was cut into height of 11 mm and length of either XX or 109 mm to fit the wells of 24-well and 6-well plate, respectively. Mass of the PDMS were measured to be 0.5611 g and 1.3179 g (n=30, standard deviation [SD] = 0.0138g, 0.0243g). For the spiking method, dimethyl sulfoxide (DMSO) was used to dissolve CP in the stock solution, and the amount of DMSO used was set to be less than 0.1% of the total exposure media.

For toxicological test using passive dosing method, PDMS loading the CP must be equipped to the exposure plate and release CP into the media. Assuming the exposure system is at equilibrium, this process may be expressed by the mass balance equation between PDMS and the media.
Here, the amount of CP released to the medium is determined by the equilibrium distribution of CP between the PDMS and the medium, which may be expressed as Eq. 3.
As can be seen in the equation, is determined by the initial concentration of CPDMS immediately prior to exposure. In turn, the desired CP concentration loaded to PDMS is determined by the partition coefficient of CP in PDMS versus in the loading solution (6:4 methanol:water). Equilibrium distribution between PDMS and the loading solution was ensured by incubation of the mixture at 20 °C for 24 hours while shaking at 150 rpm. The equilibrium was confirmed by measuring the concentration in PDMS (CPDMS,0, mg/L) and the loading solution (Cloading solution, mg/L) independently.
Finally, representing the desired CP concentration in the experiment medium can be expressed as Eq. 5 using two different equilibrium partition coefficients and given experimental conditions.
Here, KPDMSsm (6:4) is the partition coefficient of PDMS and sm (6:4), KPDMSm is the partition coefficient of PDMS and the medium, Csm is the concentration of CP applied to sm (6:4) in mg/L, and Vm and VPDMS are volumes of the medium and PDMS in mL, respectively.

Total media exposure volumes were 1 mL for the 24-well plate and 10 mL for the 6-well plate. For exposure of worms, the worms were pooled in E-tubes, and after 1~2 minutes to allow the worms to settle to the bottom, the amount of total exposure organism was determined with respect to 100 uL volume. Exposure concentration of chlorpyrifos was set to be 0.3 mg/L (water solubility of CP is XXX mg/L at 20 °C). For spiking method, stock solution was spiked into the medium containing the worm. For passive dosing method, loaded PDMS was attached to the plate and incubated for 24 hours to reach equilibrium before exposing the worms. The worms were incubated in respective medium for 24 hours at 20 °C. Exposure concentration of passive dosing method was varied from 0.3 mg/L to 0.15 mg/L, 0.06 mg/L, and 0.03 mg/L, while exposure time points were taken from beginning of the experiment until 8 hours in 2 hour intervals.

Concentration of CP within the exposure medium was re-extracted by PDMS for analysis. 1 mL of the exposure medium was transferred to a vial, and disc PDMS with diameter of XX mm was added. The vial was shaken at 150 rpm at 25 °C for 24 hours. Next, CP inside the PDMS was extracted and disc PDMS was replaced by the same amount of ethyl acetate for reverse extraction under same conditions for 24 hours. The sample was analyzed using gas chromatography, and recovery test for extraction error was conducted for 10 times for each of the three concentrations.