Cytotoxicity of phenanthrenes extracted from Aristolochia contorta in human proximal tubular epithelial cell line.
OBJECTIVE
Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is predominantly a result of aristolochic acid I (AA-I) intoxication. However, other unidentified phytotoxins have indeed been postulated as the cause of this unique interstitial nephropathy. The purpose of this study was to investigate the cytotoxicity of other phenanthrene derivatives extracted from Aristolochia contorta in the human proximal tubular epithelial cell line HK-2.


METHODS
After HK-2 cells were incubated with an indicated concentration of test compounds for 24 h, cell viability was assessed by lactate dehydrogenase (LDH) leakage assay (cell membrane damage) in combination with MTT assay (metabolic capability). Cellular morphologic assessments were performed with a phase-contrast inverted microscope and transmission electron microscope.


RESULTS
In all test compounds at 5 microg/ml, AA-I, 7-methoxy-aristololactam IV and aristololactam IVa showed cytotoxic activity in HK-2 cells in both MTT assay and LDH leakage assay (p < 0.01). At high concentration (5-80 microg/ml), these three compounds caused a dose-dependent decrease in MTT reduction and a dose-dependent increase in LDH leakage compared to non-treated cells (p <0.01). In LDH leakage assay, 40 mug/ml 7-methoxy-aristololactam IV induced a 1.58-fold LDH leakage compared to AA-I at the same concentration (p < 0.01). Moreover, the IC50 of these three compounds were 16.675 microg/ml for AA-I, 4.535 microg/ml for 7-methoxy-aristololactam IV, and 30.244 microg/ml for aristololactam IVa in MTT assay. The cellular morphologic assessments suggest interactions with cell membrane and intracellular structures such as lysosome and mitochondria are likely to be involved in cell injury induced by these three compounds.


CONCLUSIONS
The potency of cytotoxic activity of aristololactam IVa and 7-methoxy-aristololactam IV extracted from A. contorta is similar to or even stronger than that of AA-I.
Renal Division, First Hospital, Peking University, Beijing, China.
Yu-jie
YJ
Wen
Tao
T
Su
Jia-wei
JW
Tang
Cui-ying
CY
Zhang
Xuang
X
Wang
Shao-qing
SQ
Cai
Xiao-mei
XM
Li
D030022:Aristolochia / Q000737:chemistry*; D034341:Aristolochic Acids / Q000633:toxicity; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008854:Microscopy, Electron; D010616:Phenanthrenes / Q000008:administration & dosage / Q000633:toxicity*; D010936:Plant Extracts / Q000633:toxicity*; D013778:Tetrazolium Salts / Q000378:metabolism
C512137:7-methoxyaristololactam IV; D034341:Aristolochic Acids; D010616:Phenanthrenes; D010936:Plant Extracts; D013778:Tetrazolium Salts; C505882:aristololactam IVa; C000228:aristolochic acid I; D007770:L-Lactate Dehydrogenase