機車廢氣對大鼠心臟及生殖發育毒性之探討

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.