J. ENVIRON. TOXICOL. Vol. 24, No. 4, 287~292 (2009) Bisphenol A 노출과소아비만 이빛나, 신혜정 1, 나현경, 이나경, 양미희 숙명여자대학교약학대학, 1 국립의료원건강증진센터소아청소년팀 Bisphenol A Exposure and Childhood Obesity Bitna Yi, Hye Jung Shin 1, Hyunkyung Na, Na Kyung Lee and Mihi Yang Sookmyung Women s University College of Pharmacy, Seoul 140-742, Korea 1 National Medical Center, Seoul 100-799, Korea ABSTRACT Over the a few decades, the incidences of obesity and associated metabolic syndrome diseases have been increased dramatically and resulted in a global health crisis. Recent findings suggest endocrine disrupting chemicals (EDCs) as an obesogen, because they disrupt normal development and homeostatic controls over adipogenesis and energy balance in obesity. Furthermore, risk of cardiovascular disease and mortality is elevated among those who were obese during childhood. Thus, we focused on etiology of obesity in children and performed biological monitoring of bisphenol A (BPA), which is a broadly exposed EDC in environment. Study subjects were age and sex-matched obese and normal children in Seoul (N=52; age, 8.67±1.46 years). Exposure levels of BPA were analyzed with HPLC/FLD as a conjugated form in urine. As results, ranges of urinary BPA were 0~54.38 μg/g creatinine (median, 4.57 μg/g creatinine). Levels of urinary BPA were 1.7 fold higher in the obese children than those in the controls (medians of obese and control children, 7.31 and 4.25 μg/g creatinine, respectively, p=0.22). In the near future, enlarge scaled studies should be performed to confirm the risk of BPA for obesity. Key words : bisphenol A, children, obesity, obesogen, biological monitoring 서 론 전세계적으로비만은지속적인증가추세를보이고있다. 한국의경우, 국민건강영양조사에따르면성인비만율은 1998 년, 2001 년, 2005 년에걸쳐각각 26.3%, 29.6%, 31.7% 로꾸준히증가하여, 현재 30% 를넘어섰다 ( 국민건강영양조사, 2005). 또한, 성 To whom correspondence should be addressed. Tel: +82-2-2077-7179, Fax: +82-2-710-9871 E-mail: myang@sm.ac.kr 인과함께, 소아청소년에서도비만율이급증하여, 1998 년소아청소년전체의 7.2% 이었던소아청소년에서의비만인구가 2005 년에는 2 배가까이증가한 12.0% 를차지하였다 ( 국민건강영양조사, 2005). 비만은당뇨, 고지혈증, 고혈압, 심혈관계질환등을포함한성인병의대표적인원인이되기때문에위와같은비만인구의증가는심각한보건문제로떠오르고있다. 특히, 소아청소년기의비만의경우, 고지혈증, 고혈압, 탄수화물대사장애, 고지혈증등의합병증을동반할수있으며성인비만으로이행되는경우가많다. 또한또래집단의신체적놀림이나따 287
288 J. ENVIRON. TOXICOL. Vol. 24, No. 4 돌림에의해감정발달에부정적인영향을줄수있다 (Must and Strauss, 1999). 오늘날비만의증가요인을현대인의식습관, 생활환경변화에따른과잉열량섭취와운동부족이외에화학물질환경 (chemical environment) 의변화에서찾는시각이새로이등장하고있다 (Newbold et al., 2007). 또한, 화학물질의증가시기는비만인구의증가시기와겹치며, 새로운화학물질이체내의지질합성, 대사또는에너지평형에영향을미침으로써비만을일으킬가능성이제기되었다 (Baillie-Hamilton, 2002). 특히, 독성화학물질의상당부분을차지하는내분비계장애물질 (endocrine disrupting chemicals, EDCs) 은호르몬유사물질로서소량으로도체내항상성을교란시킬수있으므로, 호르몬수용체를지닌지질조직의대사를교란시킬가능성을간과할수없다 (Grun and Blumberg, 2006). 따라서, 비만에영향을미치는비만유발환경호르몬이라는의미의 오베소겐 (obesogen) 이라는신개념이등장하였고, bisphenol A (BPA), diethylstibestrol (DES), phthalates 등이이호르몬수용체신호전달계에영향을미쳐비만을유발하는 오베소겐 이라는가능성이제기되었다 (Grun and Blumberg, 2007). 또한, 여러동물실험및 in vivo 실험에서태아기또는일생동안내분비계장애물질의노출이지질대사에영향을미친다고보고된바가있다 (Rubin et al., 2001; Nikaido et al., 2004; Newbold et al., 2005). 그러나, 태아의발달단계에서는내분비계장애물질중일부만이비만을촉진할가능성이있으며, 성인에서는오히려지방합성을억제하는것으로나타나이에대한연구가더필요한실정이다 (Heindel, 2003). BPA [2,2-bis (4-hydroxyphenyl)propane, CAS# 80-05-7] 는플라스틱과에폭시수지의생산에사용되는단량체로, 오늘날식품포장재를비롯하여캔, 우유병, 치과용수지등에다양하게이용되어, 일상생활을통해쉽게노출될수있는내분비계장애물질이다. 실제본연구진이한국인을대상으로수행한 BPA 인체모니터링연구에서피험자소변중 97.5% 의시료에서 BPA 가검출된바있어, BPA 의폭넓은일반인의노출에대한건강영향이우려되고있다 (Yang et al., 2006). BPA 는에스트로겐활성을가지며생식계장애, 면역계위해, DNA adduct 형성등과관련된것으로알려져있고 (Atkinson and Roy, 1995; Kobayashi et al., 2002; Honma et al., 2002; Sawai et al., 2003) 최근에는 BPA 와비만과의관련도보고되고있다 (Baillie-Hamilton et al., 2002; Heindel et al., 2003; Newbold et al., 2008). 동물실험에서 BPA 는분화된지방세포로의당유입을촉진한바있으며 (Sakurai et al., 2004) 인슐린과함께투여할때, 섬유아세포를지방세포로분화시키는경향이보고되었다 (Masuno et al., 2002). 또한, 주산기와출산직후단계의쥐에서 BPA 의노출에의해지방조직의질량이증가되었다고보고된바있다 (Miyawaki et al., 2007). 그러나난소가제거된성인암컷쥐에서는 BPA 노출에의해체질량지수 (BMI) 의증가가현저히억제된보고도있어 (Nunez et al., 2001), 태아의발달단계와성인단계, 즉, 노출시점에서그작용의차이를보였다. 인체의경우, BPA 는지방조직과지방세포로부터비만관련질환의가능성을낮추는것으로알려진 adiponectin 의방출을억제하는것으로알려져있다 (Hugo et al., 2008). 한편, 혈청 BPA 농도가일반여성보다비만여성에서더높게나타난보고 (Takeuchi et al., 2004) 를제외하면, 비만과관련한 BPA 인체노출모니터링연구는매우부족한실정이다. 더욱이소아비만은소아가 EDC 에고감수성인구인점, 향후성인병으로의진행가능성및우리사회의성인병관련사회 경제적부담등을고려할때, 소아비만의병인으로서위험성이우려되는 EDC 의영향연구가시급한실정이다 (Bearer, 1995; Fitzgerald et al., 1998). 따라서, 본연구는 BPA 노출모니터링을통하여 BPA 노출과소아비만의상관관계를조사하였다. 재료및방법 1. 연구대상자및시료채집 피험자는서울시소재초등학교학생중건강검진에참여한소아자원자로, 소아의소변약 15 ml 를 BPA 오염이없음을확인한코니컬튜브 ( 이등, 2009) 에채집하였다. 뇨는수집후, 즉시 -80 C 에냉동상태를유지하여 center ( 숙대약대독성학교실 ) 로운반되어분석전까지 -80 C 에보관하였다. 본연구는소아와그부모로부터동의를얻었고국립의료원 IRB (Institutional Review Board) 를통과하였다. 이하 BPA 분석은성과나이를일치하고무작
December 2009 Yi et al. : Bisphenol A 노출과소아비만 289 위추출한각 26 명의정상, 비만군에서수행하였다. 2. 뇨중 BPA 분석 본연구진이확립한분석법에따라 (Yang et al., 2003) 분석대상물질인 BPA 를내부표준물질인 bisphenol B (BPB, Tokyo Chemical Industry Company, Tokyo, Japan) 와상대정량하여역상 HPLC/FLD 법으로분석하였다. 우선, 소변시료 1mL 를취해 15 ml 공전초자시험관에넣고, 이에 1 μg/ml 농도의 BPB 0.1 ml 를가하였다. 이어, 반응액에 2.0 M sodium acetate 60 μl, β-glucuronidase 48 μl (2,784 U) (Type H-1, Sigma) 를가하고진탕항온수조 (37 C, 5 시간 ) 에서배양하였다. 배양후, 2 N 염산을 200 μl 가한후, ethylacetate 5 ml 를첨가하여추출, 원심분리하여상층액 2mL 취하는액 - 액추출을 2 회반복하였다. 모아진상등액 4mL 은진공농축하여 60% acetonitrile 300 μl 에녹인후, 원심분리하여상층액을취하고그중 20 μl 를 HPLC 에주입하여 total BPA (free+conjugated form) 을정량하였다. 유리형 (free form) 의 BPA 는 β-glucuronidase 대신동량의증류수를가하여동일한과정으로분석하였다. 뇨시료의 creatinine 보정은 Ogata-Taguchi 법 (1988) 에따라 creatinine 을분석하고보정에적용하였다. 분석시스템은 dual Younglin SP930D pumps (Younglin, Seoul, Korea), MIDAS COOL autosampler (Spark Holland, Emmen, The Netherlands), Jasco FP- 2020 plus Fluorescence Detector (Jasco, Great Dunmow, UK), X-Terra C18 column (5 μm, 4.6 250 mm, Waters, Wexford, Ireland) 로구성되었다. 기울기용리법 ( 개시, 물 : acetonitrile, 75 : 25; 45 분, 45 : 55; 50 분, 75 : 25; 60 분, 75 : 25) 으로유속조건 1 ml/min, 형광여기파장 225 nm, 방출파장 305 nm 에서분석하였다. 3. 통계분석 정상아동과비만아동의성별비교에는 Fisher s Exact test 2-tail 를하였으며, 연령, 키등의비교에는 ANOVA 를하였다. 뇨중 BPA 농도와성별, 비만유무관계는 Wilcoxon test 로분석하였다. 본연구에서모든통계분석에 JMP Version 4 (SAS Institue, Cary, NC, USA) 를사용하였으며, 통계적유 의는 p 0.05 로판정하였다. 1. 피험자특성 결과및고찰 피험자의특성은 Table 1 과같다. 피험자의총인원은 52 명으로, 비만아동과정상아동의수는각각 26 명이었다. 전체성비에서남아가여아보다다소많았고, 평균나이는약 9 세였다. 평균신장에서비만아동과정상아동간에유의한차이가없었고, 다만, 체중과 BMI 에서유의적인차이가있었다. 비만기준은성별연령별체질량지수가 95 백분위수이상인경우로정의하였으며 BMI 의분포는비만아동에서는 17.5~28.4 kg/m 2 이었으며, 정상아동에서는 12.5~19.7 kg/m 2 로나타났다. Table 1. Characteristics of the subjects Item (unit) Obese (N=26) Normal (N=26) p value Sex (N (%)) Male 15 (58) 15 (58) 1.00 a Female 11 (42) 11 (42) Age (yrs) 8.65±1.49 8.69±1.46 0.93 b Height (cm) 127.82±10.09 125.12±8.19 0.29 b Weight (kg) 34.48±10.52 24.63±5.69 0.01 b BMI (kg/m 2 ) 20.61±3.10 15.53±1.75 0.01 b a Fishexr s Exact test 2-tail b ANOVA 2. 뇨중 BPA 의분포 뇨에서 BPA 를분석한결과, 검출된시료에서유리형을분석한결과, 유리형 BPA 는검출되지않았다. 따라서, 본피험자의 BPA 노출은모두 conjuated form 으로존재하며뇨중분포는 Fig. 1 과같이왼쪽으로치우치는분포를나타내어정규분포를따르지않았다. 이하 BPA level 통계분석에서는비모수통계법을수행하였다. 뇨중 BPA 농도의중간값은 4.58 μg/g creatinine (0~54.38 μg/g creatinine) 로, 2006 년본연구진에의해한국인을대상으로수행한 BPA 바이오모니터링결과 (N=172; 0.0005~243.43 μg/g creatinine; 중간값, 14.93 μg/g creatinine) 와비교해볼때 (Yang et al., 2006), 본인구집단에서 BPA 노출은다소낮은
290 J. ENVIRON. TOXICOL. Vol. 24, No. 4 3. 뇨중 BPA 농도와비만과의상관성 -10 0 10 20 30 40 50 60 Total BPA (μg/g creatinine) Fig. 1. Distribution of urinary BPA levels (μg/g creatinine): Upper part of the figure shows an outlier box plot with the square in the box showing the interquartile range: x=level of BPA; y= frequency BPA/creatinine (μg/g) 60 50 40 30 20 10 0-10 0 1 Normal, 0; Obese, 1 Fig. 2. Comparison of urinary BPA levels (μg/g creatinine) between cases and controls (P=0.22): median for cases, 7.31 μg/g creatinine; median for controls, 4.25 μg/g creatinine). 것으로추정된다. 또한, 성별에따른뇨중 BPA 농도차이는관찰되지않았다 ( 남아, 중간값, 4.75 μg/g creatinine, 여아, 중간값, 4.31 μg/g creatinine, p=0.98). BPA 의검출한계 (LOD) 는 0.4 μg/l 였으며, 통계및환산을위하여불검출시료에대하여최소값 (1.35 μg/l) 의 10 분의 1 값 (0.135 μg/l) 을적용하였다. 뇨중 BPA 농도와비만과의상관성을조사한결과, 비만아동의뇨중 BPA 농도는정상아동의것보다높은경향을보였으나 (Fig. 2), 유의적인차이는없었다 ( 비만아동, 중간값, 7.31 μg/g creatinine; 정상아동, 중간값, 4.25 μg/g creatinine, p=0.22). 이에관련하여현재까지국내외적으로참고할만한소아비만과 BPA 노출량과의상관관계에관한연구는전무한실정이다. 다만, 성인비만과 BPA 노출상관관계를살핀다른연구를살펴보면일본의비만여성 (N=19) 과정상여성 (N=7) 의혈중 BPA 를측정하여비교하였을때, 비만여성의혈중 BPA 농도가유의적으로높았다는보고가있다 ( 비만여성, 1.04± 0.09 ng/ml; 정상여성, 0.71±0.09 ng/ml: Takeuchi et al., 2004). 또한, 미국인구 (N=1,455) 를대상으로한보고에서뇨중 BPA 농도와비만도지수 (BMI) 의상관관계가유의하지않게나타나지않다고보고되었다 (Lang et al., 2008). 따라서, BPA 노출과소아비만과의상관관계, 나아가 BPA 노출이소아비만관련질환에미치는건강위해에대하여보다집중적인연구가필요하다고사료된다. 결 론 본연구진은내분비계장애물질인 BPA 의소아비만에미치는건강위해를조사하기위하여서울소재초등학교에재학중인소아를대상으로 BPA 노출모니터링을실시하고, 소아의비만여부와그상관성을분석하였다. 그결과, 전체피험자소아의뇨중 BPA 농도는중간값이 4.57 μg/g creatinine (N= 52; 0~54.38 μg/g creatinine) 이었다. 이는이전에본연구진에의해수행된 BPA 노출모니터링결과보다낮은수준이었다. 소아의비만여부와뇨중 BPA 농도와의비교에서는비만아동의뇨중 BPA 농도는정상아동의뇨중 BPA 보다다소높은경향을보였으나유의적이지않았다. 그러나, 피험자수등본연구의제한점을고려하고, 소아비만의사회적심각성, in vivo 및동물실험을통해보고되고있는 BPA 와소아비만과의상관관계등을고려할때, 향후 BPA 의건강위해종말점으로서그노출과소아비만의상관관계
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