Significant Dose-dependent Cytotoxicity Research Articles

Cellular uptake, cytotoxicity, apoptosis and DNA-binding investigations of Ru(II) complexes

Three new compounds, [Ru(Hdpa)2PyIP](ClO4)2·2H2O (1) [Ru(Hdpa)2FyIP](ClO4)2·2H2O (2) and [Ru(Hdpa)2IIP](ClO4)2·2H2O (3) have been synthesized and characterized by spectroscopic techniques such as elemental analysis, UV/Vis, FT-IR, 1H NMR, 13C NMR and mass spectra. The CT-DNA binding properties of 1–3 have been investigated by absorption, emission spectroscopy and viscosity measurements. Experimental results suggested that they can interact with DNA through intercalative mode with different binding strengths. These were found to promote the cleavage of plasmid DNA. Cell viability results indicated that all compounds showed significant dose dependent cytotoxicity in selected cell lines and 1 shown higher cytotoxicity than cisplatin on HeLa cells. Cellular uptake studies were studied by flow cytometry and confocal microscopy.

Brassinin Induces Apoptosis in PC‐3 Human Prostate Cancer Cells through the Suppression of PI3K/Akt/mTOR/S6K1 Signaling Cascades

The oncogenic PI3K/Akt/mammalian target of rapamycin (mTOR) signaling axis and its downstream effector, the ribosomal protein S6 kinase 1 (S6K1) play a key role in mediating cell survival in various tumor cells. Here, we investigated the effects of brassinin (BSN), a phytoalexin first identified as a constituent of cabbage, on the PI3K/Akt/mTOR/S6K1 activation, cellular proliferation, and apoptosis in PC-3 human prostate cancer. BSN exerted a significant dose-dependent cytotoxicity and reduced constitutive phosphorylation of Akt against androgen-independent PC-3 cells as compared to androgen-dependent LNCaP cells. Moreover, knockdown of androgen receptor (AR) by small interfering RNA enhanced the potential effect of BSN on induction of apoptosis in LNCaP cells. BSN clearly suppressed the constitutive activation of PI3K/Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, TUNEL staining, loss of mitochondrial membrane potential, down-regulation of antiapoptotic and proliferative proteins, activation of caspase-3, and cleavage of PARP. Additionally, BSN could block broad-spectrum inhibition of PI3K/Akt/mTOR/S6K1 axes, and aberrant Akt activation by pcDNA3-myr-HA-Akt1 plasmid could not prevent the observed suppressive effect of BSN on constitutive mTOR activation. Finally, overexpression of Bcl-2 also attenuated BSN-mediated apoptosis in PC-3 cells. Taken together, our findings suggest that BSN can interfere with multiple signaling cascades involved in tumorigenesis and might be provided as a potential therapeutic candidate for both the prevention and treatment of prostate cancer.

Cytotoxic activity of extracts from Hypochaeris radicata

Pasture-associated stringhalt is an acquired equine disease characterized by peripheral neuropathy and hyperflexion of the pelvic limbs. The disease occurs most commonly during periods of drought in horses grazing pastures heavily contaminated by Hypochaeris radicata. We hypothesized that stringhalt is caused by neurotoxins elaborated by H. radicata in response to the stress of drought conditions. Supernates were collected from H. radicata that were stressed (or not) by immersion in copper chloride solution, then extracted with ethyl acetate and dried. Dilutions of extracts from stressed (SE) and control, unstressed (UE) plants were incubated with myelinating spinal cord cultures (MSCC) established from fetal Swiss mice, and with spinal ganglion cultures (SGC) and dermal fibroblast cultures derived from neonatal mouse tissues. Cytotoxicity in culture monolayers was evaluated both morphologically by microscopy and by release of lactate dehydrogenase activity into culture supernates. Three different SGC preparations were exposed to a single H. radicata extract and single preparations of fibroblasts and MSCC were exposed to three different extracts. Repin, a plant-derived sesquiterpene lactone neurotoxin, was included as a positive control. Significant dose-dependent cytotoxicity was seen within 24 h in all three culture types when incubated with SE or repin. Complete morphologic destruction of culture monolayers was induced by the highest concentrations tested of SE (100 μg/mL) and repin (30 μg/mL). Cytotoxic effect of SE was significantly greater than that of UE for all three cell types and was not due to copper contamination of the extract. This study has identified a cytotoxic activity in leaf exudates of H. radicata that was upregulated by the model stressor, copper chloride.

Synthesis, interaction with DNA, cytotoxicity, cell cycle arrest and apoptotic inducing properties of ruthenium(II) molecular “light switch” complexes

In an endeavor toward the development of metal-based anticancer drugs, we present here the design, synthesis and characterization of three ruthenium(II) functionalized phenanthroline complexes with extended π-conjugation. These complexes have been shown to act as promising CT-DNA intercalators as evidenced by UV–visible, luminescence, emission quenching by [Fe(CN)6]4−, DNA competitive binding with ethidium bromide and salt dependent studies. All three complexes [Ru(Hdpa)2PPIP]2+ (1), [Ru(Hdpa)2PIP]2+ (2), [Ru(Hdpa)24HEPIP]2+ (3) clearly demonstrated that they can bind to DNA through the intercalation mode. Cell viability experiments indicated that all complexes showed significant dose dependent cytotoxicity in selected cell lines. The apoptosis and cell cycle arrest were also investigated. The complexes were docked into DNA-base-pairs using the ‘GOLD’ (Genetic Optimization for Ligand Docking), docking program.

Anti-inflammatory, Gastroprotective, and Cytotoxic Effects ofSideritis scardicaExtracts

Sideritis scardica Griseb. (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of gastrointestinal complaints, inflammation, and rheumatic disorders. This study aimed to evaluate its gastroprotective and anti-inflammatory activities. Besides, continuously increasing interest in assessing the role of the plant active constituents preventing the risk of cancer was a reason to make a detailed examination of the investigated ethanol, diethyl ether, ethyl acetate, and N-butanol extracts regarding cytotoxicity. Oral administration of the investigated extracts caused a dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. Gastroprotective activity of the extracts was investigated using an ethanol-induced acute stress ulcer in rats. The cytotoxic activity of plant extracts was assessed on PBMC, B16, and HL-60 cells and compared to the cytotoxicity of phenolic compounds identified in extracts. Apoptotic and necrotic cell death were analyzed by double staining with fluoresceinisothiocyanate (FITC)-conjugated annexin V and PI. The developed HPLC method enabled qualitative fingerprint analysis of phenolic compounds in the investigated extracts. Compared to the effect of the positive control, the anti-inflammatory drug indomethacine (4 mg/kg), which produced a 50 % decrease in inflammation, diethyl ether and N-butanol extracts exhibited about the same effect in doses of 200 and 100 mg/kg (53.6 and 48.7 %; 48.4 and 49.9 %, respectively). All investigated extracts produced dose-dependent gastroprotective activity with the efficacy comparable to that of the reference drug ranitidine. The diethyl ether extract showed significant dose-dependent cytotoxicity on B16 cells and HL-60 cells, decreasing cell growth to 51.3 % and 77.5 % of control, respectively, when used at 100 µg/mL. It seems that phenolic compounds (apigenin, luteolin, and their corresponding glycosides) are responsible for the diethyl ether extract cytotoxic effect. It also appears that induction of oxidative stress might be involved in its cytotoxicity, since B16 and HL-60 cells increased their ROS production in response to treatment with diethyl ether extract. Neither of the tested extracts nor any phenolic compounds showed significant cytotoxic effect to human PBMC. These results demonstrated the potent anti-inflammatory and gastroprotective activities, as well as the promising cytotoxicity.

Pretreatment with an Ethanolic Extract of Taiwanofungus camphoratus (Antrodia camphorata) Enhances the Cytotoxic Effects of Amphotericin B

Taiwanofungus camphoratus, a well-known Chinese medicine used in Taiwan, possesses several pharmacological functions, including anticancer effects. In the present study, we aimed to investigate a novel anticancer effect by pretreating cancer cells with an ethanolic extract of T. camphoratus (TCEE) followed by the administration of an antifungal agent amphotericin B (AmB). Both TCEE and AmB showed significant dose-dependent cytotoxicity in HT29 cells. Pretreatment with a nontoxic dose of TCEE enhanced the cytotoxicity of AmB. Furthermore, significant apoptotic cell death was found in cells treated with TCEE and AmB. A combination treatment with AmB plus TCEE resulted in a significant repression of tumor growth in HT29 xenografts. Collectively, our results indicated that combined treatment with AmB and TCEE effectively induced apoptosis and inhibited tumor growth. In the future, TCEE may serve as a potential complementary and alternative medicine to treat patients suffering from colorectal cancer.

DNA binding, cytotoxicity, and apoptotic-inducing activity of ruthenium(II) polypyridyl complex

There is considerable interest in the interactions of ruthenium (Ru)(II) complexes with DNA as well as the biological impact of the interactions. Here, by using isothermal titration calorimetry, viscosity measurement, and circular dichroism, we investigated the interactions of a new Ru(II) complex, [Ru(dmp)(2)PMIP](2+){dmp = 2,9-dimethyl-1,10-phenanthroline, PMIP = 2-(4-methylphenyl)imidazo[4,5-f]1,10-phenanthroline}, with calf thymus DNA (CT DNA). The Ru(II) polypyridyl complex and CT DNA formed a tight 1:1 complex with a binding constant of exceeding 10(6) M(-1) and with a binding mode of intercalation. Cell viability experiments indicated that the Ru(II) complex showed significant dose-dependent cytotoxicity to human lung tumor cell line A549. Further flow cytometry experiments showed that the cytotoxic Ru(II) complex induced apoptosis of human lung cancer cell line A549. Our data demonstrated that the Ru(II) polypyridyl complex binds to DNA and thereby induces apoptosis in tumor cells, suggesting that anti-tumor activity of the Ru(II) complex could be related to its interaction with DNA.

Involvement of Fatty Acid Synthase in Azacytidine-Induced Cytotoxicity in Waldenstrom's Macroglobulinemia.

Abstract 3731Poster Board III-667Azacytidine is a potent demethylation agent and has been used for the treatment of myelodysplastic syndrome. Despite a role in reactivating epigenetically silenced tumor-suppressor genes, the mechanisms of action for azacytidine are not well understood. The relationships between the reactivation of tumor-suppressor genes and the significance for clinical responses also remain to be established. Azacytidine can incorporate into both RNA and DNA strands and may therefore act through multiple pathways. We therefore investigated the therapeutic potential of azacytidine in Waldenstrom's Macroglobulinemia (WM), and characterized certain molecular changes associated with azacytidine. We found that azacytidine exhibited significant dose-dependent cytotoxicity against WM cell lines, and primary WM cells. Treatment of BCWM.1 WM cells with 2 uM of azacytidine rapidly induced cell cycle arrest at G1, and also induced apoptosis at 48 hours. Cleavage of caspase 3, 7, 8, 9 and PARP-1 suggested an involvement of mitochondrial and death receptor pathways in azacytidine-induced apoptosis. The BH3 proteins Puma and Bim, and the pro-apoptotic protein Bax were up-regulated while the anti-apoptotic proteins Bcl-2 and Bcl-xl remained unchanged after treatment with azacytidine. In addition, azacytidine did not induce Akt and NFκB pathways. To further elucidate molecular changes associated with azacytidine treatment, we performed genome-wide microarray expression in BCWM.1 WM cells treated with azacytidine. We observed that the gene encoding fatty acid synthase (FASN) was among the most down-regulated of targets. FASN has been linked to tumor cell proliferation, and its expression was observed by us to be upregulated by microarray studies in WM patients, and further validated as a highly over-expressed target by quantitative RT-PCR analysis in WM patients versus healthy donors. Moreover, by these studies we show that knockdown of FASN by small interfering RNA resulted in growth arrest, and also induced apoptosis in BCWM.1 WM cells. Cerulenin, a specific inhibitor of FASN derived from a natural product, induced strong cytotoxicity against BCWM.1 and primary WM cells at 20 uM in 24 hours. In addition, a synergistic induction of apoptosis in BCWM.1 WM cells was observed in the presence of both azacytidine and cerulenin. The results of this study support an important role for FASN in azacytidine-induced cytotoxicity in WM cells, and highlight a novel mechanistic pathway for this agent, as well as a novel target for drug therapy in WM. A clinical study investigating the activity of azacytidine in WM is planned. Disclosures:No relevant conflicts of interest to declare.

Cytotoxic aggregates of α-lactalbumin induced by unsaturated fatty acid induce apoptosis in tumor cells

The effects of three fatty acids on cytotoxic aggregate formation of Ca 2+-depleted bovine α-lactalbumin (apo-BLA) have been studied by UV absorbance spectroscopy and transmission electron microscopy. The experimental results demonstrate that two unsaturated fatty acids, oleic acid and linoleic acid, and one saturated fatty acid, stearic acid, induce the intermediate of apo-BLA at pH 4.0–4.5 to form amorphous aggregates in time- and concentration-dependent manners. These aggregates are dissolved under physiological conditions at 37 °C and further characterized by fluorescence spectroscopy, circular dichroism and time-of-flight mass spectrometry. Our data here indicate that the structural characteristics of these aggregates are similar to those of HAMLET/BAMLET ( human/ bovine α-lactalbumin made lethal to tumor cells), a complex of the partially unfolded α-lactalbumin with oleic acid. Cell viability experiments indicate the aggregates of apo-BLA induced by oleic acid and linoleic acid show significant dose-dependent cytotoxicity to human lung tumor cells of A549 but those induced by stearic acid have no toxicity to tumor cells. Furthermore, the cytotoxic aggregates of apo-BLA induced by both unsaturated fatty acids induce apoptosis of human lung cancer cell line A549, suggesting that such cytotoxic aggregates of apo-BLA could be potential antitumor drugs. The present study provides insight into the mechanism of fatty acid-dependent oligomerization and cytotoxicity of α-lactalbumin, and will be helpful in the understanding of the molecular mechanism of HAMLET/BAMLET formation.

Targeting the Bcl-2 Family of Proteins in Hodgkin Lymphoma: In Vitro Cytotoxicity, Target Modulation and Drug Combination Studies of the BH3 Mimetic ABT-737

Introduction: Currently, patients with refractory Hodgkin lymphoma (HL) or those who relapse multiple times have an extremely poor prognosis. Even patients who do not relapse often experience late toxicities, including secondary cancer, heart failure, pulmonary dysfunction, and infertility. Therefore, new agents and novel therapeutic approaches are urgently needed. Anti-apoptotic proteins such as Bcl-2 and Bcl-xl have been found to be associated with the growth and survival of Hodgkin Reed–Sternberg cells and thus carry the potential to be effective targets for therapeutics. In this study we report the in vitro cytotoxicity, biological correlative findings and drug combination analysis of the novel BH3 mimetic ABT-737 [Abbott laboratories (Abbott Park, IL)] against HL cells.Materials and Methods: HL cell lines (KMH2 and HDLM2) were cultured in the presence of increasing concentrations of ABT-737 or its enantiomer control. Normal bone marrow stromal cells were used as controls for non-specific cytotoxicity. Cell growth inhibition was measured by Alamar blue assay and the induction of apoptosis was demonstrated by an annexin specific staining technique. Time and drug concentration dependent changes in proteins involved in cell survival and apoptosis were investigated by Western blot analysis. The ability of ABT-737 to influence the anti-lymphoma activity of a panel of twenty distinct chemotherapeutic agents was evaluated by drug combination and cell growth inhibition studies. Combination indices (CI) were calculated to identify therapeutic enhancement of different agents in the presence of Bcl-2 inhibition by ABT-737.Results: ABT-737 showed significant dose-dependent cytotoxicity and apoptotic activity against HL cells with an approximate IC50 of 1mM with maximum cell death occurring at about 5mM. At the molecular level, increased levels of cleaved caspase3 and PARP, as well as annexin positivity, were noticeable within three hours of treatment with the agent. Drug combination studies have shown the ability of Bcl-2 inhibition to synergize with novel therapeutic agents that target histone deacetylase function (Apicidin, CI 0.35), Hsp90 stability (17-AAG, CI 0.3) and the activity of specific receptor tyrosine kinases (Sorafenib, CI 0.7 and Sunitinib CI 0.3 for HDLM2 and CI 0.9 for KMH2). Among conventional anti-neoplastic agents, ABT-737 showed significant synergistic activity with irinotecan and oxaliplatin (CI 0.6). Importantly, treatment with ABT-737 decreased the expression of the critical HL cell growth promoter, NF- B as determined by band densities on Western blot analysis: a two fold decrease in KMH2 and a five fold decrease in HDLM2 cells. Specific target modulation was demonstrated by changes in key apoptosis and cell survival regulators such as Bcl-XS/L, Bcl-X, p53 and survivin by two to 10 fold decreases in Western blots.Discussion: Data presented in this study support the hypothesis that Bcl-2 family of proteins can be an effective target for therapeutics in HL. We have shown that the BH3 mimetic ABT-737 induces apoptosis in these cells, characterized by the modulation of key components of cell growth and survival pathways. In addition, we have identified distinct classes of anti-lymphoma and anti-neoplastic agents whose activities are enhanced by concurrent inhibition of Bcl-2. These findings provide the rationale for further evaluation of ABT-737 and the subsequent clinical development of a targeted anti-Bcl-2 therapy for refractory Hodgkin lymphoma.

Phenotypic and Functional Differences between Human Liver Cancer Endothelial Cells and Liver Sinusoidal Endothelial Cells

Background/Aims: The phenotypic and functional characteristics of microvascular endothelial cells derived from human liver cancer (HLCEC) were analyzed in vitro and compared with those of human liver sinusoidal endothelial cells (LSEC). Methods and Results: Flow-cytometric and real-time PCR analysis indicated that expressions of tumor necrosis factor receptor (TNFR) p75, αvβ3 and αvβ5 were increased, while those of TNFR p55 and intercellular-adhesion molecule 1 (ICAM-1) were decreased in HLCEC compared with LSEC. The functional analysis indicated that HLCEC exhibited higher angiogenic ability than LSEC, including proliferation capacity, ability to form capillary-like networks and release of matrix metalloproteinases. In response to tumor necrosis factor, LSEC exhibited a significant dose-dependent cytotoxicity, while HLCEC did not. Moreover, the coagulant and fibrinolytic capacity was increased in HLCEC. In addition, tumor cell adherence was significantly higher on HLCEC than on LSEC, while leukocyte adherence was lower on HLCEC than on LSEC. The cytoadherence of HLCEC was inhibited by antibodies against αvβ3 and αvβ5,and of LSEC by antibodies against ICAM-1. Conclusion: These results indicate that tumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue. These differences are valuable for understanding tumor angiogenesis and metastasis.

Novel cytotoxic agents from an unexpected source: Bile acids and ovarian tumor apoptosis

Objective Unique biologic activities have been identified for the 4 different bile acids: cholic acid (CA, chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and ursodeoxycholic acid (UDCA). The aim of this study was to examine and compare the effects of these 4 bile acids on the human ovarian cancer cell lines A2780 and A2780-CP-R(cisplatin-resistant) and to evaluate mechanisms of action. Methods Antiproliferative effects were determined by the cytotoxic MTT assay. Cells undergoing apoptosis were identified by morphologic analysis of cells stained using Diff-Quick and nuclear staining with DAPI and by quantitative nucleosome ELISA assay. Cells were lysed in buffer after 24 h of exposure to three different concentrations of bile acid (50 mM, 200 mM, and 400 mM) and protein concentrations were determined. Cell extracts containing 25 mg of protein were assayed for protein kinase C (PKC) enzyme activity. Results None of the bile acids stimulated proliferation of ovarian cancer cells. CA and UDCA had only minimal cytotoxic effect even at maximum concentrations. In contrast, DCA and CDCA administration resulted in statistically significant dose-dependent cytotoxicity in both platinum sensitive and platinum-resistant cell lines ( p < 0.05). Cells incubated with DCA and CDCA exhibited morphologic features characteristic of apoptosis. The quantitative nucleosome ELISA assay demonstrated over 10 times increased nucleosome levels after cells were treated for 24 h by DCA and CDCA at 200 mM and 400 mM as compared to CA or UDCA treatment and to untreated controls ( p < 0.01). All 4 bile acids reduced PKC activity at concentrations of 200 and 400 mM ( p < 0.01). Conclusions CDCA and DCA have significant cytotoxic activity in ovarian cancer cells via induction of apoptosis. The mechanism of apoptosis appears to be mediated by alternative kinases distinct from PKC. CDCA and DCA may have clinical utility in the treatment of ovarian cancer pending in vivo confirmation of activity especially in cisplatin-resistant disease.

Antitumor activity of common antibiotics against superficial bladder cancer

Objectives The recurrence rate for superficial bladder tumors treated with complete resection averages 88%. Intravesical chemotherapy decreases the recurrence rate by only 14%; thus, new chemotherapeutic agents are needed. Antibiotics are often used to prevent infections after transurethral resection of bladder tumors. Oral intake of antibiotics results in significantly greater concentrations in the urine than in the serum. Our objective was to evaluate four commonly used urinary antibiotics for their cytotoxic activity against bladder cancer cells at clinically relevant concentrations. Methods Three human transitional cell carcinoma lines—HTB9 (grade 2), T24 (grade 3), and TccSup (grade 4)—were exposed to ciprofloxacin, trimethoprim-sulfamethoxazole, cefazolin, or nitrofurantoin at concentrations from 0 (control) to 1000, 1000, 5000, and 2000 μg/mL, respectively, for 96 hours. Cytotoxicity was evaluated using the MTT colorimetric assay. Six replicates were used for each data point, and the results are reported as the mean ± standard deviation. Results Significant cytotoxicity ( P <0.001) was seen, starting at 12.5 μg/mL (HTB9, TccSup) and 50 μg/mL (T24) for ciprofloxacin, 31.25 μg/mL (HTB9, TccSup) and 62.5 μg/mL (T24) for trimethoprim-sulfamethoxazole, 19.5 μg/mL (HTB9) and 156.3 μg/mL (T24, TccSup) for cefazolin, and 7.8 μg/mL (HTB9, T24, TccSup) for nitrofurantoin. Cytotoxicity was dose dependent for all four antibiotics, and the maximal effect did not differ among antibiotics. Conclusions Commonly used antibiotics exhibit significant dose-dependent cytotoxicity against bladder cancer cells at concentrations achievable in the urine after oral administration. The administration of antibiotics after transurethral resection of bladder tumors might prevent seeding of cancer cells and thereby decrease the recurrence rate. Preclinical data such as these must be considered in the design of clinical trials addressing recurrence after transurethral resection of bladder tumors.

FUNCTIONAL p53 MUTATION AS A MOLECULAR DETERMINANT OF PACLITAXEL AND GEMCITABINE SUSCEPTIBILITY IN HUMAN BLADDER CANCER

Paclitaxel and gemcitabine are promising new agents for treatment of human bladder cancer. We determine how the presence or absence of p53 function impacts the cytotoxic effects of these chemotherapeutic agents in human bladder cancer. The J82 human bladder cancer (TCC) cell line was transfected with a temperature sensitive p53 (tsp53) mutant that functions as mutated p53 at 37C but functions as wild-type (normal) p53 at 32C. Susceptibility of these inducible p53 TCC cells to paclitaxel and gemcitabine induced cytotoxicity was evaluated and kill significance determined between sub-lethal and lethal doses. Significant paclitaxel dose dependent cytotoxicity was observed in J82 TCC cells lacking normal p53 and tsp53 transfected cells at 37C, which was the mutant p53 temperature in transfectants between maximal and minimal kill concentrations for either (p <0.001). Likewise, significant cytotoxicity was observed in parental J82 TCC at 32C (p <0.001), while restoration of p53 function in tsp53 transfected cells on shift to 32C abrogated significant dose dependent cytotoxicity. Gemcitabine caused significant cell death in the cell lines incubated at either temperature and, thus, was equally effective regardless of cellular p53 function (p <0.001, respectively). Paclitaxel requires functionally mutated p53 to induce cell death in human bladder cells, indicating that it may be more effective against TCC with p53 mutations than against TCC, which lacks p53 abnormalities, while gemcitabine is effective regardless of p53 function. These findings provide a rationale for selecting chemotherapy based on the p53 status of individual bladder cancers.

Oxmetidine (H2 receptor antagonist) induced cytotoxicity in isolated rat hepatocytes.

Oxmetidine, a new and more potent analogue of the H2 receptor antagonist, cimetidine, was recently withdrawn from clinical trials because of associated hepatotoxicity. We investigated the potential hepatotoxicity of the drug in vitro and in vivo in the rat. In addition, we investigated, in in vitro experiments, the potential hepatoxicity of other gastric acid inhibitory drugs (cimetidine, ranitidine, omeprazole and nolinium bromide). In in vitro experiments, oxmetidine, at various concentrations, was added to isolated hepatocyte incubations and cytotoxicity was assayed by trypan blue exclusion. In in vivo experiments, oxmetidine was administered both i.p. and orally, and hepatotoxicity was assessed by serum biochemical measures (transaminases, alkaline phosphatase, 5' nucleotidase, gamma glutamyl transpeptidase) and liver histopathology. In the in vitro studies, the addition of oxmetidine to the hepatocyte incubations was associated with significant (P less than 0.001) dose and time dependent cytotoxicity. However, the in vivo experiments revealed no significant changes in serum biochemistry and no significant alterations in liver histopathology up to 72 h following the administration three different dosages of oxmetidine. Of the other gastric acid inhibitory drugs, only nolinium bromide was associated with significant (P less than 0.001) in vitro cytotoxicity. Our in vitro observations establish that oxmetidine is cytotoxic to isolated rat hepatocytes and suggest that nolinium bromide be further evaluated for potential hepatotoxicity.