Effects of Motorcycle Exhaust on Heart and Reproductive Development Toxicity in Rats

Abstract

暴露於品質不良的空氣、心臟疾病的好發，及男性精液品質的惡化，可能具有密切的關係。台灣都會區以機車廢氣 (motorcycle exhaust，ME) 為主要空氣汙染源。本文模擬人類可能暴露環境條件下，雌性大鼠暴露二行程機車廢氣二週，心臟重量對體重的相對重量有顯著上升，暴露 ME 組大鼠心臟的脂質過氧化現象也較控制組明顯上升，組織穀胱甘肽含量含量下降，cytochrome P450 的7-ethoxycoumarin O-dealkylase 活性下降，抗氧化酵素過氧化氫酶 (catalase，CAT) 活性下降，而雄性大鼠在相同條件暴露四週下，各生化指標亦有類似的變化趨勢，且抗氧化酵素穀胱甘肽轉移酶 (glutathione S-transferase，GST)、超氧化歧化酶 (superoxide dismutase，SOD) 及穀胱甘肽過氧化酶 (glutathione peroxidase，GPX)， 活性皆下降，表示心臟的抗氧化系統失效可能為導致氧化性傷害之一種機制。雄性大鼠在暴露八週後，心臟病理切片之結果顯示心臟左、右心室尺寸增大、心臟隔膜的心肌細胞輕微纖維化，且右心室部位有單核細胞浸潤，左心室肌纖維有輕微腫大與肌纖維間隙寬度擴大的現象。心臟超音波數據顯示，心臟瓣膜及左心室後壁厚度增厚，而左心室空腔體積明顯下降，左心室心肌肥厚等現象。雄性大鼠在相同條件下，暴露四行程機車廢氣每天分別各為二、四、六小時，發現每日暴露四、六小時組別的大鼠心臟相對重量顯著上升，但心臟的各生化指標則皆無明顯變化。　將懷孕第 7 天 (gestational day 7，GD 7) 之母鼠，呼吸暴露二行程機車廢氣持續至懷孕第 20 天 (GD 20)，懷孕母鼠於 GD 20 及 GD 21 的體重，及此其間體重增加的幅度皆顯著低於控制組與自由組。暴露組母鼠的生產子代數目 / 著床點比率亦有低於控制組和自由組的趨勢，顯示暴露組中有較多的母鼠發生早期死胎的情況。觀察暴露組子代雄、雌性仔鼠在發育過程中，體重皆有明顯低於控制組與自由組的趨勢，且 anogential distance (AGD) / 體重比率、anovaginal distance (AVD) / 體重比率及vaginal-genital distance (VGD) / 體重比率具有上升的趨勢，推測暴露組母鼠泌乳的成分，可能具有來自廢氣中的毒性物質，而導致子代仔鼠發育情形較差，且在生殖發育的過程中，因雄激素的分佈受到干擾，產生雄性化的趨勢。　雄性大鼠連續暴露二行程機車廢氣八週後，與雌性大鼠進行交配，觀察子代雄、雄性仔鼠的生殖發育情形。子代仔鼠於青春前期時，暴露組的存活率低於控制組，但不具統計意義上的顯著差異；暴露組雄性仔鼠於出生第 1 天 (postnatal day 1，PND 1) 及雌性仔鼠在 PND 14 時，肛門-生殖點距離 AGD / 體重比率均明顯低於控制組，且雌性仔鼠陰道開口的時間明顯提早。每日觀察雌性仔鼠動情週期，結果顯示，暴露組的週期規律性較差，正常的週期次數亦較少。雄、雌性仔鼠分別於 PND 56、98 與 140 時犧牲，雄性仔鼠在 PND 56 的前列腺、提肛與球海綿體肌 (LABC) 與尿道球腺，及 PND 98 時副睪與 LABC 相對重量明顯上升；暴露組雌性仔鼠於 PND 56 時卵巢相對重量有明顯上升，而在 PND 140 肝臟相對重量則顯著下降。子代雄性仔鼠睪丸的脂質過氧化、穀胱甘肽含量，以及睪丸與副睪尾的精子密度則皆沒有明顯差異。另外，探討雄、雌性子代仔鼠肝臟的生化指標的結果顯示，三個時間點暴露組雄性仔鼠的穀胱甘肽 glutathione 含量皆有下降的趨勢，其中以 PND 56 與 140 具有統計意義的顯著差異，雌性仔鼠亦皆有略低於控制組的趨勢，但皆無顯著差異。脂質過氧化方面，未加入亞鐵離子的情形下，暴露組雄性仔鼠的 molondialdehyde 值皆有下降趨勢，其中 PND56 與 140 具有顯著差異，而加入亞鐵離子後，暴露組於 PND 140 亦明顯下降；雌性仔鼠則僅在加入亞鐵離子的情形下，暴露組分別於 PND 56 及 98 有顯著下降。肝臟單氧酶方面，在 PND 98 時，暴露組雄性仔鼠aniline hydroxylase 活性有明顯下降的情形，雌性仔鼠 erythromycin N-demethylase 活性亦具有顯著的下降。探討雄性與雌性仔鼠的抗氧化酵素 GST、SOD、CAT、GPX 等活性，結果皆無明顯差異。　本研究首次證明機車排放廢氣對大鼠心臟生化與功能指標的不良影響、子代雄性與雌性仔鼠生殖發育之干擾，以及對懷孕母鼠早期死胎率、子代生殖發育的負面影響。此結果將有助於我們對機車廢氣的危害評估。The cardiac diseases and poor quality of semen are possibly associated with air pollution. Motorcycle exhaust (ME) is a major source of air pollution in the urban areas in Taiwan. The present studies showed that inhalation exposure of adult male rats to 2-stroke ME at environmental realistic conditions for 4 weeks increased heart relative weight and lipid peroxidation level, and decreased glutathione content, and the activities of 7-ethoxycoumarin O-dealkylase, glutathione S-transferase, superoxide dismutase, and glutathione peroxidase enzyme acitivities in heart. Similar effects were observed in female rats under same experimental conditions during 2 weeks exposure. The 2-stroke ME induced-cardiac oxidative damage is probably due to antioxidant defense failure. The histophathological study revealed that inhalation exposure to 2-stroke ME inhalation exposure for 8 weeks caused a slight larger size in the left and right ventricle of rat heart compared to control. Hearts showed focal minimal to slight myofibrosis in the septa, mononuclear cell infiltration in the right ventricle, and slight swelling myofibers and widening interspace between myofibers of left ventricle in ME group. The results of echocardiography analysis showed that 2-stroke ME increased the septa and left ventricle posterior wall thickness and LV mass and decreased the dimension of left ventricle cavity significantly. Under similar experimental conditions using a 4-stroke motorcycle engine of 2, 4, and 6 hours daily exposure for 4 weeks respectively, the heart relative weight were increased markedly in 4-hour and 6-hour daily exposure groups. There were no significant changes of cardiac effects in these ME groups. Inhalation exposure of pregnant rats to 2-stroke ME from gestational (GD) 7 to GD 20, the body weights of ME group at GD 20 and 21 were lower than the body weights of control and free groups significantly. ME also decreased the number of offspring/number of implantation site ratio. The results showed that ME caused more fetal deaths in early periods. We have observed adverse effects of ME on reproductive development of offspring. The body weight of offspring was lower than control and free groups for the duration of development. Furthermore, the anogential distance (AGD)/body weight, anovaginal distance (AVD)/body weight and vaginal-genital distance (VGD)/body weight ratio of ME group female offspring were altered significantly. The results revealed that the ME exposure of pregnant female rats inhibited the general growth and disturbed the reproductive development of offspring.　Adult male rats were exposed to 2-stroke ME for 8 weeks and subsequently mated with untreated female rats and the reproductive development of offspring was studied. The viability of ME group offspring showed a trend toward decrease. The AGD/body weight ratio of male and female offspring at postnatal day (PND) 1 and PND 14, respectively, were lower than control markedly. The vaginal opening day of ME group female rats was advanced significantly. The pattern of estrous cyclicity showed that the ME group female offspring had more disordered regularity. Furthermore, the number of normal cyclicity and number of normal cyclicity/rat ratio of ME group female offspring were lower than control. The male and female offspring were sacrificed at PND 56, 98, and 140 respectively. The prostate, levatro ani bulbocavernosus (LABC) and bulbourethral glands relative weights at PND 56, and epididymis and LABC relative weights at PND 98 of ME group male offspring were increased significantly. The ovary relative weight at PND 56 was increased, and liver relative weight at PND 140 of ME group female offspring was decreased markedly. Lipid peroxidation level, glutathione content, and sperm density were not altered in male offspring testis. On other experiments, we have investigated the biochemical marker changes in offspring liver. The result showed that glutathione contents were decreased in ME group male offspring liver at PND 56 and 140. Lipid peroxidation level in ME male offspring liver was lower than control at PND 56 and 98. Lipid peroxidation analysed by thiobarbituric acid-reactive substances formation with ferrous iron showed that the level of ME group female offspring in liver was lower than control at PND 140. With respective to monooxygenase activities, aniline hydroxylase activity of male offspring and erythromycin N-demethylase activity in female offspring liver were decreased significantly. Antioxidant enzyme activities were not changed.　To the best of our knowledge, this is the first to show adverse effects of exposure to ME on biochemical markers and function of heart, reproductive development of offspring, and fetal viability. These results may assist in assessing the health risk associated with ME exposure.目 錄 中文摘要…………………...…………………………………………………….............1英文摘要…………………………………………………………………………............4一章 前言一節、日趨嚴重的空氣汙染議題…………………………….…………………..7二節、空氣汙染導致的健康危害及其機制……………….……………………..7 第三節、目前台灣機動車使用情況及機車廢氣組成分…………….…………......8 第四節、空氣汙染與心臟疾病之間的關聯…………………………………….....10五節、空氣汙染與精液品質惡化以及不孕之間的關聯…………………….....10六節、本篇研究目的………………………………………………………….....11二章　研究方法與步驟 第一節、呼吸暴露實驗 Ⅰ. 實驗動物……………………………………………………………………13 Ⅱ. 機車廢氣暴露裝置系統…………………………………………………....13 Ⅲ. 大鼠呼吸暴露機車廢氣實驗……………………………………………....14 Ⅳ. 懷孕母鼠呼吸暴露機車廢氣實驗………………………………………....14 第二節、 精子毒性評估 Ⅰ. 睪丸及副睪精子密度計算…………..……………………………………..14 第三節、 呼吸二行程機車廢氣大鼠之懷孕率實驗……...……….………………….15 第四節、 子代仔鼠發育觀察……………..…………………………………………...15 第五節、 酵素實驗測試 Ⅰ. 細胞微粒體及細胞質之製備……………………………………………....16. 蛋白濃度測定……………………………………………………………....16. 單氧酶活性測試 a. 7-Ethoxycoumarin O-Dealkylase Activity Assay………………………….....17 b. 7-Ethoxyresorufin O-Deethylase Activity Assay……………….………….......17 c. Methoxyresorufin O-Demethylase Activity Assay…………....……………….17 d. Pentoxyresorufin O-Dealkylase Activity Assay……………………………….17 e. Erythromycin N-Demethylase Activity Assay………………...………………18 f. Aniline Hydroxylase Activity Assay…………………………...……………...18 Ⅳ. 抗氧化酵素活性測試 a. Glutathione S-Transferase Activity Assay…………………………………….18 b. Superoxide Dismutase Activity Assay…………………………………….......18 c. Catalase Activity Assay…………………………………………………….....19 d. Total Glutathione Content Assay……...………………………………….......19 第六節、 脂質過氧化分析……………...…………………………………………….19 第七節、 血清酵素分析 Ⅰ. 肝臟細胞損傷指標. Alanine Aminotransferase Activity Assay………………………………….....19. Aspartate Aminotransferase Activity Assay………………...……………..20. 心肌細胞損傷指標. Lactate Dehydrogenase Activity Assay………………………………….....20. Creatine Phosphokinase Activity Assay………………………………………20八節、 大鼠心臟功能測定. 心電圖測定…………………………………………………………………..21. 心臟超音波測定…………………………………………………………......22九節、 數據統計分析………….………………………………………………….22三章 結果一節、 二行程機車廢氣對雄性及雌性大鼠器官相對重量之影響………….....23二節、 暴露二行程機車廢氣之雄性大鼠心臟超音波及心電圖變化之影…………………………..…………………………………………….......23第三節、 暴露二行程機車廢氣之雄性大鼠心臟組織病理變化之影響…………….24四節、 二行程機車廢氣對雄性及雌性大鼠血清肝臟細胞及心肌細胞急性傷生物指標之影響…….…………………………………………...........25 第五節、 二行程機車廢氣對雄性及雌性大鼠心臟穀胱甘肽含量和脂質過氧化影響…………………….…………………………………………...........25 第六節、 二行程機車廢氣對雄性及雌性大鼠心臟單氧酶活性與抗氧化酵素活之影響…………...……...…………………………………………...........26七節、 四行程機車廢氣對雄性大鼠體重、心臟相對重量及各項生化指標 之影響…………………….……………………………...............................26八節、 懷孕母鼠暴露二行程機車廢氣期間體重變化與早期死胎率之影 響..………………………...…………………………………………….......27九節、 暴露二行程機車廢氣之懷孕母鼠對子代仔鼠體重、AGD/體重比率、 AVD/體重比率及 VGD/體重比率與動情週期規律性之影響…...............28第十節、 二行程機車廢氣對雄性大鼠精子密度變化之影響……....………..…….29十一節、 二行程機車廢氣對雄性大鼠生殖能力之影響…….……………..….….29十二節、 暴露二行程機車廢氣之親代雄性大鼠對子代仔鼠青春前期的存活 率、體重與 AGD/體重比率之影響………….………………...……….29十三節、 暴露二行程機車廢氣之親代雄性大鼠對子代雄性仔鼠陰莖包皮分離、雌性仔鼠陰道開口與初次動情時間之影響………..…......…….30十四節、 暴露二行程機車廢氣之親代雄性大鼠對子代仔鼠青春期及成年生殖發育指標之影響………………………………………...…….....30十五節、 暴露二行程機車廢氣之親代雄性大鼠對子代雄性仔鼠睪丸精子度、穀胱甘肽含量與脂質過氧化之影響…………............................31十六節、 暴露二行程機車廢氣之親代雄性大鼠對子代雌性仔鼠動情週期律之影響……...………………………………………………………32十七節、 暴露二行程機車廢氣之親代雄性大鼠對子代雌、雄性仔鼠肝臟胱甘肽含量、脂質過氧化、單氧酶活性與抗氧化酵素活性之響…………………………………...………………………………...33四章　討論一節、 二行程機車廢氣影響心臟生化指標及造成心肌肥厚的可能機制…….35二節、 暴露二行程機車廢氣懷孕母鼠與早期死胎、子代仔鼠生長不良生殖發育情形影響之關聯……………………….…….……………...39三節、 親代雄性大鼠暴露二行程機車廢氣影響子代仔鼠生殖發育的可 能機制……….…………………………………………..….…………….41四節、 親代雄性大鼠暴露二行程機車廢氣影響子代仔鼠肝臟單氧酶活與性賀爾蒙的關聯……….……………………………….…………...43五節、 結論……………………………………………………………………….45考文獻………………………………………………………………………………….46目錄able 1. Effects of 2-stroke ME inhalation exposure on relative organ weights in male rats……………………………………...………………………………..56able 2. Effects of 2-stroke ME inhalation exposure on relative organ weights in female rats………………...………………...…………………………………57able 3. Effect of treatment with 2-stroke ME inhalation exposure on echocardiography in male rats………………...…………………………………58able 4. Effect of treatment with 2-stroke ME inhalation exposure on electrocardiography n male rats…………………………………...…………......................................59able 5. Effects of treatment with 2-stroke ME inhalation exposure on aspartate aminotransferase and alanine aminotransferase enzyme activities in male rat serum………………………………………...…………………………………...60able 6. Effects of treatment with 2-stroke ME inhalation exposure on lactate dehydrogenase and creatine kinase enzyme activities in male and female rat sera…………………………………………...…………………………………..61able 7. Effects of treatment with 2-stroke ME inhalation exposure on total glutathione content and lipid peroxidation in male and female rat hearts...……..62able 8. Effects of treatment with 2-stroke ME inhalation exposure on monooxygenase activities in male and female rat hearts………………...……..63able 9. Effects of treatment with 2-stroke ME inhalation exposure on antioxidant enzyme ctivities in male and female rat hearts………………………………………….64able 10. Effects of treatment with 4-stroke ME inhalation exposure on body weights and relative organ weights of heart in male rats……...………………………...65able 11. Effects of treatment with 4-stroke ME inhalation exposure on lipid peroxidation, total GSH content, monooxygenase and antioxidant enzyme activities in male rat heart and cardiac cell damage markers n male rat serum…..…………………………...…………….………………….66able 12. Effects of treatment of pregnant female rats with 2-stroke ME inhalation xposure on body weight……………………………….……………………….67able 13. Effects of treatment of pregnant female rats with 2-stroke ME inhalation exposure on number of offspring / number of implantation site ratio…....68able 14. Effect of in utero treatment with 2-stroke ME inhalation exposure on body weight in male offspring….………….………………………………...69able 15. Effect of in utero treatment with 2-stroke ME inhalation exposure onody weight in female offspringing………………………………………….....70able 16. Effects of in utero treatment with 2-stroke ME inhalation exposure on strus cyclicity regularity and he frequency of estrus cyclicity phases in emale offspring……………………………………………………………...…71able 17. Effect of 2-stroke ME inhalation exposure on sperm density in male rats…..….72able 18. Reproductive toxicity of male rats exposed to 2-stroke ME by inhalation……..73able 19. Effects of paternal treatment with 2-stroke ME inhalation exposure of parental male and female rats on pre-puberty male offspring viability,ody weight, and AGD/body weight ratio………………………………….…..74able 20. Effects of paternal treatment with 2-stroke ME inhalation exposure on the age of preputial separation in male offspring and age of vaginal opening and onset of estrus cycle in female offspring….…...………………….………...........…...75able 21. Effects of paternal treatment with 2-stroke ME inhalation exposure of onhe pubertal and adult male offspring reproductive development…….…….…...76able 22. Effects of paternal treatment with 2-stroke ME inhalation exposure on the pubertal and adult female offspring reproductive development……….…...…..77able 23. Effects of paternal treatment with 2-stroke ME inhalation exposure on ody weight and relative organ weight of the pubertal and adult male ffspring……..…………...……………………………………………………..78able 24. Effects of paternal treatment with 2-stroke ME inhalation exposure on ody weight and relative organ weight of the pubertal and adult female ffspring………………………………………………………………………..79able 25. Effects of paternal treatment with 2-stroke ME inhalation exposure on sperm densities in male offspring testis and cauda epididymis…….………….80able 26. Effects of paternal treatment with 2-stroke ME inhalation exposure on otal glutathione content and lipid peroxidation in male offspring testis…….…81able 27. Effects of paternal treatment with 2-stroke ME inhalation exposure on strus cyclicity regularity in female offspring………………………………….82able 28. Effects of paternal treatment with 2-stroke ME inhalation exposure on he frequency of estrus cyclicity phases in female offspring…………………...83able 29. Effects of paternal treatment with 2-stroke ME inhalation exposure on otal glutathione content and lipid peroxidation in male and female ffspring liver…………………………………………………………………..84able 30. Effects of paternal treatment with 2-stroke ME inhalation exposure on onooxygenase activities in male and female rat livers……………………….85able 31. Effects of paternal treatment with 2-stroke ME inhalation exposure on ntioxidant enzyme activities in male and female offspring livers…………….86目錄ig 1. Effect of treatment with 2-stroke ME inhalation exposure on echocardiographyn male rats………………...……………………………………………………...87ig 2. Effects of 2-stroke ME inhalation exposure on cardiac histopathological hanges in male rats……...……………………………………………………….88ig 3. Effects of 2-stroke ME inhalation exposure on cardiac histopathological hanges in male rats (continued)…………………………….…….89 (continued)ig 4. Effects of 2-stroke ME inhalation exposure on cardiac histopathological hanges in male rats (continued)……………….…………………….90 (continued)ig 5. Effects of 2-stroke ME inhalation exposure on cardiac histopathological hanges in male rats (continued)….…………….……………………91 (continued)ig 6. Effects of 2-stroke ME inhalation exposure on cardiac histopathological hanges in male rats (continued)……………….……………...……..92 (continued)ig 7. Effect of in utero treatment with 2-stroke ME inhalation exposure on GD/body ratio in male offspring……………….…….………………………….93ig 8. Effect of in utero treatment with 2-stroke ME inhalation exposure on GD/body ratio in female offspring…………………..…………………………..94ig 9. Effect of in utero treatment with 2-stroke ME inhalation exposure on VD/body ratio in female offspring…………..……………..……………………95ig 10. Effect of in utero treatment with 2-stroke ME inhalation exposure on GD/body ratio in female offspring…………..…….………………...…………...96ig 11. Effect of in utero treatment with 2-stroke ME inhalation exposure on estrus yclicity regularity in female offspring……………………………….……………97ig 12. Effect of paternal treatment with 2-stroke ME inhalation exposure on estrus yclicity regularity in female offspring (continued).…..………..….....98 (continued