Nemoto N, Sakurai J, Tazawa A, Ishikawa T
Department of Experimental Pathology, Cancer Institute, Tokyo, Japan.
Cancer Res. 1989 Nov 1;49(21):5863-9.
Induction of aryl hydrocarbon hydroxylase (AHH) was studied in mouse hepatocytes in primary culture and compared with that in rat hepatocytes. Enzyme activity in hepatocytes from C57BL/6 mice was found to increase dose dependently after treatment with benz(a)anthracene. However, the induction was strictly dependent on culture medium. Although appreciable levels of AHH activity were inducible in Sprague-Dawley rat hepatocytes cultivated in either Dulbecco's minimal essential medium (DMEM) or Waymouth's medium, C57BL/6 mouse cells cultivated in DMEM responded to the inducer only very slightly, whereas those in Waymouth's or Ham's F-12 medium demonstrated a marked increase. Proline, but not glutamic acid or cysteine, all of which were lacking in DMEM but present in Waymouth's and Ham's F-12 medium, restored the potential for response to the cells in DMEM. While increased amounts of P450 mRNA in C57BL/6 cells cultivated in DMEM were transient and decreased after a peak observed at 24 h, levels of mRNA in Waymouth continued to demonstrate an increase at 48 h. Addition of proline to mouse hepatocytes in DMEM increased the generation of transcripts without, however, influencing the decrease observed from 24 h to 48 h. Timing of treatment with benz(a)anthracene and incubation in Waymouth greatly influenced the eventual AHH activity. Thus, while enzyme activities measured at 48 h were in the same range after treatment with benz(a)anthracene for either the whole period or only for the initial 24 h, and prominent induction was observed with cells in Waymouth for 24 to 48 h regardless of whether they were at first cultivated in DMEM or Waymouth, levels remained low if the cells were incubated in DMEM during the 24- to 48-h period. These observations suggest that induction of AHH in mouse hepatocytes is regulated by both transcriptional and posttranscriptional events and that proline-dependent events are required for expression of the enzyme activity.
在原代培养的小鼠肝细胞中研究了芳烃羟化酶（AHH）的诱导情况，并与大鼠肝细胞中的诱导情况进行了比较。发现用苯并(a)蒽处理后，C57BL/6小鼠肝细胞中的酶活性呈剂量依赖性增加。然而，这种诱导严格依赖于培养基。虽然在杜尔贝科改良伊格尔培养基（DMEM）或韦茅斯培养基中培养的斯普拉格-道利大鼠肝细胞中可诱导出相当水平的AHH活性，但在DMEM中培养的C57BL/6小鼠细胞对诱导剂的反应非常轻微，而在韦茅斯培养基或哈姆F-12培养基中的细胞则表现出明显增加。脯氨酸可恢复DMEM中细胞对诱导剂的反应能力，而谷氨酸或半胱氨酸则不能，DMEM中缺乏这两种氨基酸，但韦茅斯培养基和哈姆F-12培养基中存在。虽然在DMEM中培养的C57BL/6细胞中P450 mRNA的增加量是短暂的，在24小时观察到峰值后下降，但韦茅斯培养基中的mRNA水平在48小时仍持续增加。向DMEM中的小鼠肝细胞添加脯氨酸可增加转录本的产生，但不影响24小时至48小时观察到的下降。苯并(a)蒽的处理时间和在韦茅斯培养基中的孵育对最终的AHH活性有很大影响。因此，虽然在整个时间段或仅在最初24小时用苯并(a)蒽处理后，48小时测得的酶活性在相同范围内，并且无论最初是在DMEM还是韦茅斯培养基中培养，在24至48小时内韦茅斯培养基中的细胞都观察到显著诱导，但如果在24至48小时期间将细胞在DMEM中孵育，活性水平仍然很低。这些观察结果表明，小鼠肝细胞中AHH的诱导受转录和转录后事件的调节，并且脯氨酸依赖性事件是酶活性表达所必需的。
