Malaveille C, Kuroki T, Brun G, Hautefeuille A, Camus A M, Bartsch H
Mutat Res. 1979 Dec;63(2):245-58. doi: 10.1016/0027-5107(79)90057-5.
In plate assays in the presence of S. typhimurium TA100 and various amounts of liver 9000 X g supernatant (S9) from either untreated, phenobarbitone- (PB) or Aroclor-treated rats, the S9 concentration required for optimal mutagenicity of aflatoxin B1 (AFB) depended both on the source of S9 and on the concentration of the test compound. In these assays, the water-soluble procarcinogen, dimethylnitrosamine (DMN) was mutagenic in S. typhimurium TA1530 only in the presence of a 35-fold higher concentration of liver S9 from PB-treated rats than that required for AFB, a lipophilic compound. In liquid assays, a biphasic relationship was observed in the mutagenicities in S. typhimurium TA100 of benzo[a]pyrene (BP) and AFB and the concentration of liver S9. For optimal mutagenesis of BP, the concentration of liver S9 from rats treated with methylcholanthrene (MC) was 4.4% (v/v); for AFB it was 2.2% (v/v) liver S9 from either Aroclor-treated or untreated rats. At higher concentrations of S9 the mutagenicity of BP and of AFB was related inversely to the amount of S9 per assay. The effect of Aroclor treatment on the microsomemediated mutagenicity of AFB was assay-dependent: in the liquid assay, AFB mutagenicity was decreased, whereas in the plate assay it did not change or was increased. As virtually no bacteria-bound microsomes were detected by electron microscopy, after the bacteria had been incubated in a medium containing 1-34% (v/v) MC-treated rat-liver S9, it is concluded that, in mutagenicity assays, mutagenic metabolites generated by microsomal enzymes from certain pro-carcinogens have to diffuse through the assay medium before reaching the bacteria. Thus the mutagenicity of BP was dependent on both the concentration of rat-liver microsomes and that of total cytosolic proteins and other soluble nucleophiles such as glutathione. At a concentration of 4.4% (v/v) liver S9, the mutagenicity of BP was about 3.6 times higher than in assays containing a 4-fold higher concentration of cytosolic fraction. Studies on the glutathione-dependent reduction of BP mutagenicity in plate assays has shown that, in the presence of liver S9 concentrations greater than that required for optimal mutagenicity, the reduction in mutagenicity was related directly to the concentration of liver S9. Thus, in the Salmonella/microsome assay, when the concentration of rat-liver S9 was increased over and above the amount required for the optimal mutagenicity of BP, the mutagenic metabolites of BP were inactivated (by being trapped with cytosolic nucleophiles and/or by enzymic conjugation with glutathione); this effect increased more rapidly than their rate of formation. The concentration of liver S9 for optimal mutagenicity of test compounds requiring activation catalyzed by mono-oxygenases seems, therefore, to be related to the departure from linearity of the relationship between the rate of formation of mutagenic metabolites and the concentration of liver S9.
在平板试验中，存在鼠伤寒沙门氏菌TA100以及来自未处理、苯巴比妥（PB）或多氯联苯处理大鼠的不同量肝脏9000×g上清液（S9）时，黄曲霉毒素B1（AFB）达到最佳诱变性所需的S9浓度既取决于S9的来源，也取决于受试化合物的浓度。在这些试验中，水溶性前致癌物二甲基亚硝胺（DMN）仅在存在来自PB处理大鼠的肝脏S9浓度比亲脂性化合物AFB所需浓度高35倍时，才在鼠伤寒沙门氏菌TA1530中具有诱变性。在液体试验中，观察到苯并[a]芘（BP）和AFB在鼠伤寒沙门氏菌TA100中的诱变性与肝脏S9浓度呈双相关系。对于BP的最佳诱变，来自经甲基胆蒽（MC）处理大鼠的肝脏S9浓度为4.4%（v/v）；对于AFB，它是来自多氯联苯处理或未处理大鼠的2.2%（v/v）肝脏S9。在更高的S9浓度下，BP和AFB的诱变性与每次试验中S9的量呈负相关。多氯联苯处理对AFB微粒体介导的诱变性的影响取决于试验：在液体试验中，AFB诱变性降低，而在平板试验中它没有变化或增加。由于在电子显微镜下几乎未检测到与细菌结合的微粒体，在细菌于含有1 - 34%（v/v）MC处理大鼠肝脏S9的培养基中孵育后，得出结论，在诱变性试验中，某些前致癌物的微粒体酶产生的诱变代谢物在到达细菌之前必须扩散穿过试验培养基。因此BP的诱变性既取决于大鼠肝脏微粒体的浓度，也取决于总胞质蛋白和其他可溶性亲核试剂如谷胱甘肽浓度。在肝脏S9浓度为4.4%（v/v）时，BP的诱变性比含有高4倍胞质部分浓度的试验高约3.6倍。在平板试验中关于谷胱甘肽依赖性降低BP诱变性的研究表明，在存在大于最佳诱变性所需肝脏S9浓度时，诱变性的降低与肝脏S9浓度直接相关。因此，在沙门氏菌/微粒体试验中，当大鼠肝脏S9浓度增加到超过BP最佳诱变性所需量时，BP的诱变代谢物被灭活（通过被胞质亲核试剂捕获和/或通过与谷胱甘肽的酶促结合）；这种作用比它们的形成速率增加得更快。因此，对于需要单加氧酶催化活化的受试化合物达到最佳诱变性所需的肝脏S9浓度，似乎与诱变代谢物形成速率和肝脏S9浓度之间关系偏离线性有关。
