飼糧中添加石蓴和中國半葉馬尾藻對肉雞砷代謝物、生物轉移及砷暴露風險評估

Abstract

砷 (arsenic, As) 生物毒性與其化學形式有關，無機砷 (Inorganic arsenic, iAs) 的毒性是有機砷的100倍以上，被國際癌症研究機構 (international agency for research on cancer, IARC) 列為第1類致癌物。海藻作為飼料原料可促進動物生長及免疫調節，但是海藻的砷累積能力是周圍海水濃度的100倍。本研究目的為建立雞肉中不同砷型態的檢驗方法，探討飼糧中添加石蓴及馬尾藻在雞肉的砷蓄積、型態及代謝模式，並評估消費者攝入含砷海藻飼料所生產的雞胸肉，無機砷的暴露風險。最後，建立砷從飼料到雞肉的生物轉移模式，並利用該模式評估商業飼料到雞肉的無機砷蓄積以及對人體砷暴露風險。 臺灣的雞肉消費量逐年增加，對雞肉中砷的形態及蓄積研究非常的少。我們建立了雞肉砷型態的分析方法，一次可檢驗雞肉中可能出現的9種形態的砷，包括無機砷 (arsenite, As(III) and arsenate, As(V))、甲基砷 (monomethylarsenic acid, MMA and dimethylarsenic acid, DMA)、有機砷 (arsenobetaine, AsB and arsenocholine, AsC) 及有機的含砷動物用藥及代謝物 (Roxarsone (Rox), p-arsanilic acid (p-ASA), and 4-hydroxyphenylarsonic acid (4-HPAA)。利用超聲處理與菠蘿蛋白酶 (bromelain) 輔助萃取，經由HPLC結合ICPMS檢測可於22分鐘內完成9種不同型態砷的分析。9種砷形態的方法定量極限 (LOQ) 為0.90 - 1.98g/kg，回收率為78.5%-109.4%，該分析方法可用於雞肉中砷形態的鑑定。 為了探討砷從飼料到雞肉的生物轉移、蓄積及代謝，將240隻一日齡肉雞分配到5個處理組，分別為基礎日糧 (對照組)，2%和5%馬尾藻 (Sargassum hemiphyllum var. chinense, SHC) 添加組、2%和5%的石蓴 (Ulva lactuca, UL) 添加組。試驗為期35天，分析飼料和雞胸肉的砷形態以及砷代謝相關酵素。結果顯示餵飼5% UL及5% SHC飼料的總砷分別比對照基礎日糧組多1.4及78倍。SHC飼料中主要砷型態為砷酸鹽 (arsenate, As(V))，而餵飼SHC的飼料所生產的雞胸肉主要砷性型態為二甲基砷酸 (dimethylarsenic acid, DMA)。而UL飼料主要砷型態為砷酸鹽 (As(V)) 和砷甜菜鹼 (arsenobetaine, AsB)，飼餵含UL飼料所生產的雞胸肉的主要代謝物為AsB。飼糧中添加SHC可增加肝臟S-腺苷甲硫氨酸 (S-adenosyl-methionine, SAM) 和砷甲基轉移酶 (arsenic methyltransferase, As3MT)，而飼餵UL可提高腎臟中砷甲基轉移酶。飼糧中添加SHC和UL對雞隻砷代謝的調節模式不同，但餵飼SHC會增加雞胸肉無機砷的積累。 海藻是動物飼料中常見的營養添加物，但也可能是砷污染的來源。針對雞隻餵飼海藻飼料所生產的雞肉進行砷的暴露風險分析，評估成人和兒童攝食添加5% SHC和5% UL的飼料所生產的雞胸肉的砷暴露風險。利用蒙地卡羅模擬估算臺灣民眾每日攝食量 (estimated daily intake, EDI) 結果顯示，兒童食用5% SHC和5% UL飼料所生產的雞肉，每日攝入無機砷的含量分別比成人高1.8和1.7倍。當暴露限值 (margin of exposure, %MOE) 為100%時，攝入高砷SHC組雞胸肉之成年消費者每公斤體重的消費率 (CR/bw) 為0.4 g/kg day (第25個百分位數，P25)，遠低於對照組及低砷UL組 (1.6 g/kg day，第85個百分位數，P85)。攝入餵養 5% SHC 飼料生產的雞胸肉的致癌風險高於餵養 5% UL 的雞胸肉，且兒童的暴露風險高於成人消費者。 生物轉移因子 (Biotransfer factor, BTF) 可評估飼料有害物質轉移到畜產品之含量。本試驗利用BTF建立砷從飼料到雞肉的生物轉移模式，BTF表示為雞肉總砷 (tAs) 或無機砷 (iAs) 含量 (g/kg) 與飼料中tAs的日攝入量 (µg/day) (tAs/tAs或iAs/tAs) 之比例。使用SHC和UL餵飼的雞飼料tAs的日攝入量及雞肉的tAs或iAs的濃度進行線性回歸分析，估算飼料tAs到全雞iAs (iAs/tAs) 的BTF為0.016 day/kg (R2 = 0.9886) 以及飼料tAs到全雞tAs (tAs/tAs) 的BTF為0.54 day/kg (R2 = 0.9939)。為了驗證BTF可以合理的估算從飼料tAs到雞肉的iAs含量，並開發用商業飼料估算對人類來自雞肉貢獻的砷暴露風險。我們進行商業飼料採樣並分析總砷含量 (n = 79)，利用BTF估算雞肉中無機砷的濃度以及隨後的暴露風險。雙變量蒙特卡羅 (Bivariate Monte Carlo) 模擬結果顯示無機砷每日攝入量 (EDI) 的第95個百分位數 (P95) 為0.002 µg/kg bw/day，低於增加0.5%肺癌發生率之基準劑量下限 (Benchmark Dose Low, BMDL0.5) 3.0 µg/kg bw/day。 綜上所述，建立了一種高通量方法同時檢測可能出現在動物產品中的九種砷形態。應用該方法分析砷從海藻飼料到雞肉的轉移，SHC海藻作為飼料原料增加砷在雞胸肉中的積累，含有SHC和UL的飼料分別通過增加肝和腎As3MT甲基化，導致雞胸肉中As(V) 轉化為DMA。然而，因為無機砷可能會在雞胸肉中積累，並增加幼兒暴露無機砷的風險，所以家禽飼料應謹慎使用含SHC的產品。此外，建立了評估從商業飼料預測雞肉砷蓄積量及砷暴露風險評估方法，餵飼商業飼料所生產之雞肉產品，攝入其雞肉來源的砷對臺灣人群的健康影響較低。

The chemical and biological toxicity of arsenic (As) dependents on its chemical forms, whereas inorganic arsenic (iAs) is more than 100 times more toxic than organic arsenic. The iAs is classified as group 1 carcinogen by the International Agency for Research on Cancer. Seaweed plays a potential role in livestock feed as an effector for immunomodulation and growth, and it may also be a potential source of As contamination. Chicken consumption in Taiwan increases yearly, and little is known about the speciation of arsenic in chicken. The purpose of this study is to develop a novel detection method for arsenic (As) species in chicken by high-performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICPMS). The effect of Ulva lactuca (UL) or Sargassum hemiphyllum var. chinense (SHC) supplementation on the accumulation and metabolites of As in broiler breasts were investigated by the method established in this study. Next, the potential health risks of iAs to consumers who ingested the breast of chicken fed with arsenic-containing seaweed were estimated. Finally, the arsenic accumulation from commercial feed to chicken meat and the arsenic exposure risk in humans was accessed by the biotransfer factor. Part I: Chicken consumption in Taiwan increases yearly, and little is known about the speciation of arsenic in chicken. A novel method was established to detect arsenic species in animal products, including inorganic arsenic (iAs) (arsenite, As(III) and arsenate, As(V)), methyl arsenic (monomethylarsenic acid (MMA) and dimethylarsenic acid (DMA)), organic arsenic (arsenobetaine (AsB) and arsenocholine (AsC)) and organic arsenic-containing animal medicines and metabolites (Roxarsone (Rox), p-arsanilic acid (p-ASA), and 4-hydroxyphenylarsonic acid (4-HPAA)). After ultrasonication combined with bromelain-assisted extraction, the analysis of nine arsenic species was completed within 22 min by HPLC combined with ICPMS. The limit of quantification of this method were in the range of 1.01 - 1.96 µg/kg and obtained recoveries were 78.5% - 109.4%. This analytical method was applied to the identification of arsenic species in chicken meats and the assessment of human arsenic exposure. Part II: In order to investigate the transfer, accumulation, and metabolism of arsenic species from feed to chicken, a total of 240 day-old broilers were randomly allocated to five treatments groups including control the group (basal diet), 2%, and 5% Sargassum hemiphyllum var. chinense (SHC), 2% and 5% Ulva lactuca (UL) supplementation group, respectively. Arsenic species in feed and chicken breast and arsenic-related metabolic enzymes were analyzed. Broilers fed 5% UL or 5% SHC ingested 1.4- or 78- fold greater total As than birds fed the control diet. The majority of As species were As(V) in the SHC feed and DMA in the breasts of chicks fed the SHC-containing diet. AsB and As(V) were the dominant metabolites in the UL-containing feed, and AsB was the major metabolite in breasts of chicks fed the UL-containing diet. Feeding SHC enhanced hepatic S-adenosyl-methionine and arsenic methyltransferase, whereas feeding UL elevated renal arsenic methyltransferase. The regulatory pattern of As metabolism is different between SHC- and UL-containing feed; SHC-supplemented feed caused increased inorganic arsenic accumulation in the chicken. Part III: This study evaluated a risk analysis that assessed the risk of arsenic exposure in adults and children consuming chicken breast produced from feed supplemented with seaweed. The Monte Carlo in the estimated daily intake (EDI) simulation results showed that the EDI of iAs was 1.8 and 1.7 times higher in children than that in adults who ate chicken supplemented with 5% SHC and 5% UL, respectively. When the margin of exposure (MOE) was 100%, the consumption rate per body weight (CR/bw) of adult consumers ingesting SHC-chicken breast was 0.4 g/kg day, which was much lower than that in the low-arsenic UL group (1.6 g/kg day). The MOE risk was generally higher in children than adults consumers, and the carcinogenic risk of ingesting chicken breast produced by feeding 5% SHC feed was higher than that feeding with 5% UL. Part IV: This study developed a viable commercial-feed risk assessment method for iAs in chicken meat. The reliable biotransfer factor (BTF) was estimated as 0.016 day/kg, as iAs/tAs (R2 = 0.9886) or 0.54 day/kg as tAs/tAs (R2 = 0.9939) for whole chicken meat. Bivariate Monte Carlo simulations (n = 10,000) indicated that the 95th percentile of EDI for iAs was 0.002 μg/kg bw/day, which was below the benchmark dose lower limit of 3.0 μg/kg bw/day. The health impact of arsenic hazards on the Taiwanese population was low for chicken meat produced from commercial chicken feeds in this study. In conclusion, A high-throughput method was developed to simultaneously detect nine arsenic species that may occur in animal products. Seaweed as a feed ingredient enhanced As accumulation in chicken breasts. The SHC-containing feed caused the conversion of As(V) to DMA in chicken breasts. However, the uses of SHC in poultry diets should be cautiously because of the potential accumulation of inorganic As species in chicken breast and the higher risk of exposure to iAs in young children. In addition, we developed a viable commercial-feed risk assessment method for iAs in chicken meat and found that Taiwanese commercial chicken feeds had low health concerns in the general Taiwanese population.