KLF4 functions as an oncogene in promoting cancer stem cell-like characteristics in osteosarcoma cells.

Despite more effective chemotherapy combined with limb-salvage surgery for the osteosarcoma treatment, survival rates for osteosarcoma patients have stagnated over the past three decades due to the poor prognosis. Osteosarcoma cancer stem cells (OSCs) are responsible for the growth and metastasis of osteosarcoma. The existence of OSCs offers a theoretical explanation for therapeutic failures including tumor recurrence, metastasis, and drug resistance. Understanding the pathways that regulate properties of OSCs may shed light on mechanisms that lead to osteosarcoma and suggest better modes of treatment. In this study, we showed that the expression level of Kruppel-like factor 4 (KLF4) is highly associated with human osteosarcoma cancer stemness. KLF4-overexpressed osteosarcoma cells displayed characteristics of OSCs: increased sphere-forming potential, enhanced levels of stemness-associated genes, great chemoresistance to adriamycin and CDDP, as well as more metastasis potential. Inversely, KLF4 knockdown could reduce colony formation in vitro and inhibit tumorigenesis in vivo, supporting an oncogenic role for KLF4 in osteosarcoma pathogenesis. Furthermore, KLF4 was shown to activate the p38 MAPK signaling pathway to promote cancer stemness. Altogether, our studies uncover an essential role for KLF4 in regulation of OSCs and identify KLF4-p38 MAPK axis as a potential therapeutic target for osteosarcoma treatment.

The regioselective glucuronidation of morphine by dimerized human UGT2B7, 1A1, 1A9 and their allelic variants.

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7 is expressed mostly in the human liver, lung and kidney and can transfer endogenous glucuronide group into its substrate and impact the pharmacological effects of several drugs such as estriol, AZT and morphine. UGT2B7 and its allelic variants can dimerize with the homologous enzymes UGT1A1 and UGT1A9, as well as their allelic variants, and then change their enzymatic activities in the process of substrate catalysis. The current study was designed to identify this mechanism using morphine as the substrate of UGT2B7. Single-recombinant allozymes, including UGT2B7*1 (wild type), UGT2B7*71S (A71S, 211G>T), UGT2B7*2 (H268Y, 802C>T), UGT2B7*5 (D398N, 1192G>A), and double-recombinant allozymes formed by the dimerization of UGT1A9*1 (wild type), UGT1A9*2 (C3Y, 8G>A), UGT1A9*3 (M33T, 98T>C), UGT1A9*5 (D256N, 766G>A), UGT1A1 (wild type) with its splice variant UGT1A1b were established and incubated with morphine in vitro. Each sample was analyzed with HPLC-MS/MS. All enzyme kinetic parameters were then measured and analyzed. From the results, the production ratio of its aberrant metabolism and subsequent metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), changes regioselectively. Double-recombinant allozymes exhibit stronger enzymatic activity catalyzing morphine than the single-recombinant alloyzymes. Compared to UGT2B7*1, UGT2B7*2 singles or doubles have lower Km values for M3G and M6G, whereas UGT2B7*5 allozymes perform opposite effects. The double allozymes of UGT1A9*2 or UGT1A9*5 with UGT2B7 tend to produce M6G. Interestingly, the majority of single or double allozymes significantly reduce the ratio of M3G to M6G. The UGT1A9*2-UGT2B7*1 double enzyme has the lowest M3G:M6G ratio, reflecting that more M6G would form in morphine glucuronide metabolism. This study demonstrates that UGT2B7 common SNPs and their dimers with UGT1A1 and UGT1A9 and their allelic variants can regioselectively affect the generation of two metabolites of morphine via altering the CLint ratios of M3G to M6G. These results may predict the effectiveness of morphine antinociception in individualized opioid treatment.

[Personalized dosing from perspective of pharmacogenomics of drug metabolizing enzymes and transporters].

Pharmacogenomics is defined as research into the relationship between inherited genetic variations in drug metabolizing enzymes, transporters and targets and individual variations in person's response to drugs (fate of drug in human body, safety and efficacy). Personalized dosing is pharmacogenomics-based therapeutic regimen tailored to other individual characteristics. This article summarizes the progress in clinical application of personalized dosing from the perspective of pharmacogenomics of drug metabolizing enzymes and transporters, and proposes to draw attention to key scientific issues (e.g., the effect of multi-genes and non-genetic factors on drug effects, the integration of therapeutic drug monitoring and pharmacogenomics); meanwhile, bottle necks in the clinical application and corresponding strategies are proposed.

[Induction study of CYP3A2 in male rats by zolmitriptan].

In our preliminary studies, we observed zolmitriptan (ZOL) treatment led to induction of CYP3A2 in male not female rats. To figure out the reason is of great significance for drug-drug interactions and personalized administration. Since growth hormone (GH) is known as the major mechanistic determinant of sexually-dimorphic gene expression like CYP3A2 in rat liver, the impacts of ZOL on both plasma GH levels in non monosodium glutamate (MSG)-treated rats and CYP3A2 expression in GH depleted MSG-treated rats were studied. ZOL was shown to partially suppress GH levels in both genders. Furthermore, CYP3A2 protein and mRNA level declined in male not female MSG-treated rats. In order to study the possible molecular events involved in the depression of GH and gender-selective induction on rat CYP3A2 by ZOL, the mRNA and protein level(whole protein and nuclear protein) of hepatocyte nuclear factor 4α (HNF4α) was investigated. Nuclear accumulation of HNF4α was observed in the normal male not female rat liver tissue following ZOL treatment. However, this kind of nuclear translocation did not occur in rat hepatocytes and MSG-treated rats. These findings demonstrated CYP3A2 inducibility by ZOL was gender-selective. GH and HNF4α may play an important role in CYP3A2 induction.

Stereoselectivity of chiral drug transport: a focus on enantiomer-transporter interaction.

Drug transporters and drug metabolism enzymes govern drug absorption, distribution, metabolism and elimination. Many literature works presenting important aspects related to stereochemistry of drug metabolism are available. However, there is very little literature on stereoselectivity of chiral drug transport and enantiomer-transporter interaction. In recent years, the experimental research within this field showed good momentum. Herein, an up-to-date review on this topic was presented. Breast Cancer Resistance Protein (BCRP), Multidrug Resistance Proteins (MRP), P-glycoprotein (P-gp), Organic Anion Transporters (OATs), Organic Anion Transporting Polypeptides (OATPs), Organic Cation Transporters (OCTs), Peptide Transport Proteins (PepTs), Human Proton-Coupled Folate Transporter (PCFT) and Multidrug and Toxic Extrusion Proteins (MATEs), have been reported to exhibit either positive or negative enantio-selective substrate recognition. The approaches utilized to study chirality in enantiomer-transporter interaction include inhibition experiments of specific transporters in cell models (e.g. Caco-2 cells), transport study using drug resistance cell lines or transgenic cell lines expressing transporters in wild type or variant, the use of transporter knockout mice, pharmacokinetics association of single nucleotide polymorphism in transporters, pharmacokinetic interaction study of racemate in the presence of specific transporter inhibitor or inducer, molecule cellular membrane affinity chromatography and pharmacophore modeling. Enantiomer-enantiomer interactions exist in chiral transport. The strength and/or enantiomeric preference of stereoselectivity may be species or tissue-specific, concentration-dependent and transporter family member-dependent. Modulation of specific drug transporter by pure enantiomers might exhibit opposite stereoselectivity. Further studies with integrated approaches will open up new horizons in stereochemistry of pharmacokinetics.

[Simultaneous determination of madecassoside, asiaticoside and their aglycones in Centella asiatica (L.) urban extracts by RP-HPLC].

OBJECTIVE: To establish a HPLC method for simultaneous determination of four major constituents (madecassoside, asiaticoside, madecassic acid and asiatic acid) in Centella asiatica (L.) urban extracts. METHODS: The analysis was performed on an Agilent 1100 HPLC system with a ZORBAX Eclipse XDB-C8 column (4.6 mm×150 mm, 5µm). The four major constituents were separated with gradient mobile phase that consists of 1mmol/L potassium dihydrogen phosphate and acetonitrile at the detection wavelength of 205 nm. RESULTS: The four major constituents all had good linear response in the determination ranges (R(2)≥0.9998). The average recoveries (n=9) were 97.4%, 93.7%, 97.5% and 99.8% with RSDs of 3.4%, 1.4%, 4.7% and 4.4%, respectively. CONCLUSION: The developed method is sensitive and has good reproducibility, which can be used as a reference for quality control of Centella asiatica (L.) urban extracts.

[Expression of human glutathione S-transferase A1, P1 and T1 in Escherichia coli].

OBJECTIVE: To construct the vectors of human glutathione S-transferase A1 (GSTA1), P1 (GSTP1), T1(GSTT1) genes and express in Escherichia coli (E. coli). METHODS: Human GSTA1, GSTP1 and GSTT1 gene whole length cDNAs were amplified by RT-PCR and then subcloned into pET-28a(+) vectors. The proteins were expressed in E. coli BL21(DE3). After purified by Ni2+ affinity chromatography, the enzymatic activities of GSTs were measured with 1-chloro-2,4 -dinitrobenzene (CDNB) as substrate. RESULTS: The correct GSTA1, GSTP1 and GSTT1 genes were cloned. And soluble GSTA1, GSTT1, GSTP1 proteins were expressed in E.coli. After purification, GSTA1, GSTT1 and GSTP1 showed good enzymatic activities, which were 17.55, 0.02, 18.75 µmol·min-1·mg-1, respectively. CONCLUSION: The expression plasmids for GSTA1, GSTT1 and GSTP1 have been constructed and the recombinant proteins are expressed successfully.

[Determination of enantiomeric impurity of timolol maleate in bulk substances and eye drops].

OBJECTIVE: To determine the enantiomeric impurity contents of domestic timolol maleate in bulk drugs and eye drops. METHODS: Enantiomer impurity of timolol was assayed by chiral high performance liquid chromatography. The chromatographic conditions were as follows:chiralcel OD chiral column (4.6 mm ×150 mm, 5µm), detection wavelength:297 nm, mobile phase:hexane-isopropanol-diethylamine (480:20:1), column temperature:25 ℃, flow rate:1.0 ml/min, sample injection volume:5 µl. RESULTS: The resolution between R- and S-timolol was more than 4. The enantiomeric impurity contents were less than 0.67% on average in two batches of timolol maleate bulk drugs, and 0.31% on average in three batches of timolol maleate eye drops. CONCLUSION: Enantiomeric impurity contents in each batch of products all meet European Pharmacopoeia criteria, which can be used as references in Chinese Pharmacopoeia criteria.

[Enantiomeric separation and impurity determination of valaciclovir hydrochloride].

OBJECTIVE: To determine the contents of L-enantiomer impurity in valaciclovir hydrochloride. METHODS: Valaciclovir enantiomers were separated and determined by using chiral high performance liquid chromatography. Chromatographic conditions were as follows:CROWNPAK(®) CR(+) chiral column (4 mm×150 mm, 5 µm), detection wavelength:254 nm, mobile phase:water-methanol-perchloric acid (19:1:0.1), flow rate:0.75 ml/min, sample injection volume:10 µl. RESULTS: D-valaciclovir was completely separated from L-enantiomer impurity. The contents of L-enantiomer impurity were 0.65%-2.62% on average in 8 batches of valaciclovir hydrochloride. CONCLUSION: Enantiomeric impurity contents in each batch of products were all meet criteria of United States Pharmacopeia, which can be used in criteria of Chinese Pharmacopeia as references.

Stereoselective binding of chiral drugs to plasma proteins.

Chiral drugs show distinct biochemical and pharmacological behaviors in the human body. The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity, which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles. In this review, the stereoselective binding of chiral drugs to human serum albumin (HSA), α1-acid glycoprotein (AGP) and lipoprotein, three most important proteins in human plasma, are detailed. Furthermore, the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed. Apart from the stereoselectivity of enantiomer-protein binding, enantiomer-enantiomer interactions that may induce allosteric effects are also described. Additionally, the techniques and methods used to determine drug-protein binding parameters are briefly reviewed.

[Establishment of in vitro evaluation model for CYP2B6 induction and its application to screen inducers among TCMs].

This paper is to report the development of a high-throughput in vitro system to screen hPXR/CAR mediated CYP2B6 drug inducers, and the application of it into the quick determination of induction activity toward CYP2B6 by various commonly used traditional Chinese medicines (TCMs) extract. Dual reporter gene assays were performed. The hPXR/CAR expression vectors and the reporter vector pGL3-CYP2B6-Luc involved in the distal and proximal promoters of CYP2B6 were co-transfected into HepG2 cells. Relative luciferase activities in cell lysate were analyzed after 48 h treatment of blank vehicle or drugs to determine the induction activity toward CYP2B6 by various commonly used TCMs extract. The positive hPXR/hCAR activators rifampicin and CITCO were applied to make sure that the reporter gene model was successfully established. Then 5 kinds of commonly used TCM extracts and 1 herbal compound were successfully investigated, some were found to activate hPXR or hCAR and therefore have the potential to induce CYP2B6 enzyme. This is the first domestic article to report the hCAR3-mediated CYP2B6 induction model and the establishment of a reporter gene system for hPXR/CAR-mediated CYP2B6 induction can be an effective and systemic in vitro method to investigate the drug inducers of CYP2B6 and to explain the mechanism involved.

Stereoselective binding of mexiletine and ketoprofen enantiomers with human serum albumin domains.

AIM: To investigate the stereoselective binding of mexiletine or ketoprofen enantiomers with different recombinant domains of human serum albumin (HSA). METHODS: Three domains (HSA DOM I, II and III) were expressed in Pichia pastoris GS115 cells. Blue Sepharose 6 Fast Flow was employed to purify the recombinant HSA domains. The binding properties of the standard ligands, digitoxin, phenylbutazone and diazepam, and the chiral drugs to HSA domains were investigated using ultrafiltration. The concentrations of the standard ligands, ketoprofen and mexiletine were analyzed with HPLC. RESULTS: The recombinant HSA domains were highly purified as shown by SDS-PAGE and Western blotting analyses. The standard HSA ligands digitoxin, phenylbutazone and diazepam selectively binds to DOM I, DOM II and DOM III, respectively. For the chiral drugs, R-ketoprofen showed a higher binding affinity toward DOM III than S-ketoprofen, whereas S-mexiletine bound to DOM II with a greater affinity than R-mexiletine. CONCLUSION: The results demonstrate that HSA DOM III possesses the chiral recognition ability for the ketoprofen enantiomers, whereas HSA DOM II possesses that for the mexiletine enantiomers.

[CYP2D6*1, CYP2D6*10 co-expressed with CYPOR in Bac-to-Bac expression system and activity determination].

CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D6*1/CYP2D6*10 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D6*1, pFastBac-CYP2D6*10 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6*1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.

[The role of ADME evaluation in translation research of innovative drug].

New Chemical Entities (NCEs) development is a systematic long-term project that involves multiple disciplines. The translation research will help to build an advanced R&D system from the basic laboratory research, preclinical studies and clinical evaluation to clinical application of drug, for the purpose of shortening the R&D cycle and accelerate the launch of new drugs. In new drug R&D and its clinical application, drug disposition (absorption, distribution, metabolism, excretion, ADME) properties are important criteria for assessing drug-likeness of candidates. ADME evaluation of NCEs plays an important role in the translation research throughout innovative drug R&D process. Therefore, ADME evaluation at the early stage of drug design and development will be helpful to improve the success rate and reduce costs, and further access to safe, effective drugs.

[Determination of quercetin metabolism in UGT1A3 cDNA-expressing cells by RP-HPLC].

OBJECTIVE: To develop a RP-HPLC method for the determination of quercetin in UGT1A3 cDNA-transfected cells. METHODS: The lysate of cells transfected with human recombinant uridine 5-diphosphate glucuronosyltransferases UGT1A3 cDNA was co-incubated with quercetin, the reaction was terminated with acetonitrile, and luteolin was used as internal standard. The determination was performed on a C(1) reversed phase column with a mobile phase of methanol-0.1% formic acid (V/V) at a flow rate of 1.0 ml/min. The gradient elution was as follows: 0 - 25 min (30:70-80:20, methanol:0.1% formic acid), > 25-25.5 min (80:20), >25.5-27 min (80:20-30:70), > 27-30 min (30:70). A UV-VIS detector was operated at 368 nm. RESULT: The standard curve was linear over the concentration range of 5-200 µmol/L (r = 0.9999). The limit of detection was 1.25 µmol/L(S/N ≥ 3), and the limit of quantification was 5 µmol/L (S/N >10, RSD = 6.99%). The method afforded recoveries of 99.1%-103.5%, and precisions for inter- and intra-assay were < 2.5% and < 8%, respectively. In addition, kinetic analysis indicated that the K(m), V(max) and CL(int) (V(max)/K(m)) values for quercetin glucuronide were (62.95 ± 13.16) µ mol/L, (284.50 ± 24.35)nmol*min⁻¹*g⁻¹ and 4.52 ml*min⁻¹*g⁻¹, respectively. CONCLUSION: The method established is accurate and simple and suitable for the determination of quercetin in UGT1A3 cDNA-expressed cells.

[Chiral separation of fluvastatin enantiomers with in vitro cellular method].

OBJECTIVE: To establish a chiral separation method for determination of fluvastatin enantiomer with in vitro cellular model. METHODS: The determination was performed on Chiralpak AD column (4.6 mm × 250 mm); and the phase consisted of hexane-isopropanol-trifluoroacetic acid (90:10:0.1) at a flow rate of 0.5 ml/min with UV detection of 239 nm. RESULT: The standard curve was linear over the concentration range of 20 µmol/L-300 µmol/L (r² = 0.9993, r² = 0.9997). The recovery for this assay was (99.4 ± 0.8)%, precision for inter-assay and intra-assay was <10 %. CONCLUSION: The normal-phase HPLC chiral separation method was accurate and suitable for study on the stereoselectivity of fluvastatin with in vitro cellular model.

[Induction of rat hepatic cytochrome P-450 3A by zolmitriptan mediated with pregnane X receptor].

OBJECTIVE: To investigate whether pregnane X receptor (PXR) is involved in the induction of rat hepatic cytochrome P450 3A1/2 by zolmitriptan. METHODS: The induction effects of zolmitriptan were investigated in three concentrations (100 micromol L(-1)), 50 micromolL(-1)) and 10 micromol L(-1))) using luciferase report gene method based on PXR. Pregnenolone-16 alpha-carbonitrile (PCN) was used as the positive control and the solvent DMSO as the negative control. RESULT: Compared with the negative groups, the positive and high level zolmitriptan groups exhibited a significant induction effect on CYP 3A1, the activity increased to 1.93 and 1.96 times, respectively (P<0.05). The positive, middle level and low level zolmitriptan groups exhibited a significant induction effect on CYP 3A2, the activity increase to 2.51, 2.10 and 1.63 times, respectively (P<0.01, <0.05 ). CONCLUSION: Zolmitriptan can significantly induce CYP 3A2 in low and middle concentrations and induce CYP 3A1 in high concentration.PXR is involved in the induction of CYP 3A1/2 by zolmitriptan.

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro.

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

Brain-derived neurotrophic factor involved epigenetic repression of UGT2B7 in colorectal carcinoma: A mechanism to alter morphine glucuronidation in tumor.

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7, as one of significant drug enzymes, is responsible on the glucuronidation of abundant endobiotics or xenobiotics. We here report that it is markedly repressed in the tumor tissues of colorectal carcinoma (CRC) patients. Accordingly, morphine in CRC cells will stimulate the expression of its main metabolic enzyme, UGT2B7 during tolerance generation by activating the positive signals in histone 3, especially for trimethylated lysine 27 (H3K4Me3) and acetylated lysine 4 (H3K27Ac). Further study reveals that brain-derived neutrophilic factor (BDNF), a secretory neurotrophin, enriched in CRC can interact and inhibit UGT2B7 by primarily blocking the positive signals of H3K4Me3 as well as activating H3K27Ac on the promoter region of UGT2B7. Meanwhile, BDNF repression attributes to the sensitizations of main core factors in poly-comb repressive complex (PRC) 1 rather than PRC2 as the reason of the depression of SUZ12 in the later complex. Besides that, the productions of two main morphine glucuronides are both increased in the BDNF deficient or TSA and BIX-01294 treated morphine tolerance-like HCT-116 cells. On the same condition, active metabolite, morphine-6-glucuronide (M6G) was accumulated more than inactive M3G. Our findings imply that enzymatic activity enhancement and substrate regioselective catalysis alteration of UGT2B7 may release morphine tolerance under the cure of tumor-induced pain.

Mechanism for ginkgolic acid (15 : 1)-induced MDCK cell necrosis: Mitochondria and lysosomes damages and cell cycle arrest.

Ginkgolic acids (GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA (15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA (15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA (15 : 1) on MDCK cells displayed a time- and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA (15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential (ΔΨm). In propidium iodide (PI) staining analysis, GA (15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA (15 : 1) induced renal toxicity.