Management of Oxidative Stress by Heme Oxygenase-1 in Cisplatin-induced Toxicity in Renal Tubular Cells

Induction of heme oxygenase-1 (HO-1) may serve as an immediate protective response during treatment with the cytostatic drug cisplatin (CDDP). Oxidative pathways participate in the characteristic nephrotoxicity of CDDP. In the present study, cultured tubular cells (LLC-PK1) were used to investigate whether induction of HO provided protection against CDDP by maintaining the cellular redox balance. The antioxidants, &#102 -tocopherol (TOCO) and N -acetylcysteine (NAC), were used to demonstrate that elevation of ROS levels contribute to the development of CDDP-induced cytotoxicity. Chemical modulators of HO activity were used to investigate the role of HO herein. Hemin was used to specifically induce HO-1, while exposure of the cells to tin-protoporphyrin (SnPP) was shown to inhibit HO activity. Hemin treatment prior to CDDP-exposure significantly decreased the generation of ROS to control levels, while inhibition of HO increased the ROS levels beyond the levels measured in cells treated with CDDP alone. Furthermore, HO induction protected significantly against the cytotoxicity of CDDP, although this protection was limited. Similar results were obtained when the cells were preincubated with TOCO, suggesting that mechanisms other than impairment of the redox ratio are important in CDDP-induced loss of cell viability in vitro. In addition, SnPP treatment exacerbated the oxidative response and cytotoxicity of CDDP, especially at low CDDP concentrations. We therefore conclude that HO is able to directly limit the CDDP-induced oxidative stress response and thus serves as safeguard of the cellular redox balance.     

Role of nitric oxide in human pulmonary microvascular endothelial cell adhesion

Cyclopentenylcytosine (CPEC) is cytotoxic to HT-29 cells in vitro and in vivo. Treatment with CPEC resulted in sensitizing HT-29 cells to cisplatin (CDDP), as evidenced by synergistic cytotoxicity. CPEC exhibits potent cytotoxicity to HT-29 cells in vitro, 2 and 24 h exposure providing an LC50 of 2.4 and 0.46 microM, respectively. Exposure of HT-29 cells to CDDP for 2 h resulted in an LC50 of 26 microM. Treatment of HT-29 cells with 1.0 or 1.25 microM CPEC and then incubating with CDDP showed synergistic cytotoxicity. Lesser synergy at very high concentrations of CPEC was demonstrated when HT-29 cells were first exposed to CDDP and then incubated with CPEC. Combination index calculations showed synergistic cytotoxicity in HT-29 cells when CPEC was combined with CDDP. Synergistic antitumor activity was demonstrable in vivo in mice transplanted with HT-29 tumor when treated with a combination of CPEC and CDDP without undue toxicity, since no excessive loss in mouse body weight or overt pathology was observed. CPEC had no influence on the total DNA adduct formation and CDDP did not affect the intracellular levels of CPEC or its metabolites, suggesting that enhanced CDDP cytotoxicity resulted from a step subsequent to excision of platinum-cross-linked DNA. These studies support a new approach for augmenting cytotoxic effect of CPEC with CDDP in treating human colon carcinoma.     

MK2206 Enhances Cisplatin-Induced Cytotoxicity and Apoptosis in Testicular Cancer Through Akt Signaling Pathway Inhibition

OBJECTIVE: To improve conventional chemotherapeutic efficacy, it is significant to identify novel molecular markers for chemosensitivity as well as possible molecules accelerating cell-killing mechanisms. In this study, we attempted to elucidate how MK2206, an allosteric Akt inhibitor, enhances the cisplatin (CDDP)-induced cytotoxicity and apoptosis in testicular cancer. MATERIALS AND METHODS: We checked three testicular cancer cell lines for the expression of phospho(p)-Akt and its downstream molecules targets by Western blot. The potential antitumor effects were analyzed by MTT assay in vitro and by subcutaneous xenograft models in vivo. The cell invasion was analyzed by transwell invasion assay, and the activities of Akt signaling pathway and expression of apoptosis-related proteins were measured by Western blot. RESULTS: Our results indicated that there was overactivation of p-Akt and its downstream molecules in testicular cancer cell lines compared with normal testis epithelium cells. MK2206 (600 nM) inhibited cell invasion in TCAM-2 and P19 cell lines and significantly increased the susceptibility of testicular cancer to CDDP. Combined with CDDP, MK2206 potentiated CDDP-induced cytotoxicity and apoptosis, with repressed expression of p-Akt and its downstream targets. The subcutaneous xenograft models also showed that a combined CDDP/MK2206 therapy completely suppressed tumor growth without any side effects. CONCLUSION: These results suggested that the concomitant use of MK2206 could enhance the CDDP-induced cytotoxicity and apoptosis in testicular cancer with the suppressed expression of Akt pathway.     

Management of oxidative stress by heme oxygenase-1 in cisplatin-induced toxicity in renal tubular cells

Induction of heme oxygenase-1 (HO-1) may serve as an immediate protective response during treatment with the cytostatic drug cisplatin (CDDP). Oxidative pathways participate in the characteristic nephrotoxicity of CDDP. In the present study, cultured tubular cells (LLC-PK1) were used to investigate whether induction of HO provided protection against CDDP by maintaining the cellular redox balance. The antioxidants, alpha-tocopherol (TOCO) and N-acetylcysteine (NAC), were used to demonstrate that elevation of ROS levels contribute to the development of CDDP-induced cytotoxicity. Chemical modulators of HO activity were used to investigate the role of HO herein. Hemin was used to specifically induce HO-1, while exposure of the cells to tin-protoporphyrin (SnPP) was shown to inhibit HO activity. Hemin treatment prior to CDDP-exposure significantly decreased the generation of ROS to control levels, while inhibition of HO increased the ROS levels beyond the levels measured in cells treated with CDDP alone. Furthermore, HO induction protected significantly against the cytotoxicity of CDDP, although this protection was limited. Similar results were obtained when the cells were preincubated with TOCO, suggesting that mechanisms other than impairment of the redox ratio are important in CDDP-induced loss of cell viability in vitro. In addition, SnPP treatment exacerbated the oxidative response and cytotoxicity of CDDP, especially at low CDDP concentrations. We therefore conclude that HO is able to directly limit the CDDP-induced oxidative stress response and thus serves as safeguard of the cellular redox balance.     

In vitro studies on genotoxicity and cytotoxicity of the anticancer drugs cisplatin and cofplaton, a caffeine-8-ether plus cisplatinum compound.

The cytotoxic and genotoxic properties of the newly designed anticancer drug 'cofplaton' were investigated. Since cofplaton is a cisplatin (CDDP) plus caffeine compound, the widely applied anticancer drug CDDP alone or in combination with caffeine was studied in parallel. As measured by the MTT test the cytotoxicity of the two drugs was comparable, but cofplaton exhibited significantly fewer genotoxic side effects than CDDP in the chromosome aberration test as well as in the SCE assay. First results from animal studies indicate that cofplaton exerts antitumor activity comparable to CDDP. Because of its relatively low genotoxicity, cofplaton seems to be a promising drug in human anticancer therapy.     

Vanadium(III)-L-cysteine protects cisplatin-induced nephropathy through activation of Nrf2/HO-1 pathway

Cisplatin (CDDP) is one of the first-line anticancer drugs; however, the major limitation of CDDP therapy is development of nephrotoxicity (25–35% cases), whose precise mechanism mainly involves oxidative stress, inflammation and cell death. Therefore, in search of a potential chemoprotectant, an organovanadium complex, viz., vanadium(III)-L-cysteine (VC-III) was evaluated against CDDP-induced nephropathy in mice. CDDP was administered intraperitoneally (5?mg/kg b.w.) and VC-III was given by oral gavage (1?mg/kg b.w.) in concomitant and pre-treatment schedule. The results showed that VC-III administration reduced (p?<?0.001) serum creatinine and blood urea nitrogen levels, suggesting amelioration of renal dysfunction. VC-III treatment also significantly (p?<?0.001) prevented CDDP-induced generation of reactive oxygen species, reactive nitrogen species, and onset of lipid peroxidation in kidney tissues of the experimental mice. In addition, VC-III also substantially (p?<?0.001) restored CDDP-induced depleted activities of the renal antioxidant enzymes such as, superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, and glutathione (reduced) level. Furthermore, histopathological study also confirmed the renoprotective efficacy of VC-III. Western blotting analysis appended by immunohistochemical data showed that VC-III treatment quite effectively reduced the expression of proinflammatory mediators such as, NFκβ, COX-2 and IL-6. VC-III administration also stimulated Nrf2-mediated antioxidant defense system by promotion of downstream antioxidant enzymes, such as HO-1. Moreover, treatment with VC-III significantly (p?<?0.001) enhanced CDDP-mediated cytotoxicity in MCF-7 and NCI-H520 human cancer cell lines. Thus, VC-III can serve as a suitable chemoprotectant and increase the therapeutic window of CDDP in cancer patients.     

Scavenging activity of indole compounds against cisplatin-induced reactive oxygen species

The antioxidant effects of indole compounds such as melatonin (MLT), tryptophan, and serotonin, on cisplatin (cis-diaminedichloroplatinum, or CDDP)-induced reactive oxygen species (ROS) generation were examined by electron spin resonance (ESR). In addition, DNA fragmentation by CDDP-induced ROS and the effect of MLT on it were analyzed in primary cultures of rat renal tubular epithelial cells. MLT and serotonin had scavenging effects on CDDP-induced hydroxy radicals (*OH), and the scavenging activity of MLT was higher than that of serotonin. The exposure of primary-cultured renal tubular cells to CDDP caused severe cytotoxicity. Tryptophan, serotonin, and 6-OH-MLT did not reduce the CDDP-induced cytotoxicity, whereas MLT did. CDDP exposure induced DNA fragmentation in primary-cultured renal tubular cells, but the simultaneous administration of MLT inhibited the DNA fragmentation. These results indicate that MLT inhibits CDDP-induced cytotoxicity by directly scavenging *OH, and that MLT markedly reduces renal cytotoxicity and DNA fragmentation caused by CDDP-induced ROS in vitro.     

Copper deprivation modulates CTR1 and CUP1 expression and enhances cisplatin cytotoxicity in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been established as a model system for cancer studies, due to the widely conserved family of genes involved in cell cycle progression, proliferation and apoptosis. In the current study, we sought to determine whether copper deprivation modulates sensitivity of yeast to cisplatin. Yeast cultures grown in low copper medium and exposed to bathocuproiene disulfate (BCS) resulted in significant reduction of intracellular copper. We report here that low copper medium rendered BY4741 hypersensitive to cisplatin (CDDP). Yeast grown in low copper medium exhibited ～2.0 fold enhanced cytotoxicity in survival and colony-forming ability, compared to copper adequate control cells grown in YPD. The effect of copper restriction on CDDP sensitivity appeared to be associated with the up regulation of CTR1, facilitating enhanced uptake and accumulation of CDDP. Also, CDDP further lowered copper deprivation-induced changes in CUP1 metallothionein levels, SOD activity and GSH levels. These changes were associated with increased protein oxidation and lipid peroxidation induced by CDDP. These results thus suggest that cisplatin cytotoxicity is potentiated under low copper conditions due to enhanced uptake and accumulation of cisplatin and also in part due to lowered antioxidant defense and increased oxidative stress imposed by copper deprivation.     

Zoledronic Acid Produces Combinatory Anti-Tumor Effects with Cisplatin on Mesothelioma by Increasing p53 Expression Levels

We examined anti-tumor effects of zoledronic acid (ZOL), one of the bisphosphonates agents clinically used for preventing loss of bone mass, on human mesothelioma cells bearing the wild-type p53 gene. ZOL-treated cells showed activation of caspase-3/7, -8 and -9, and increased sub-G1 phase fractions. A combinatory use of ZOL and cisplatin (CDDP), one of the first-line anti-cancer agents for mesothelioma, synergistically or additively produced the cytotoxicity on mesothelioma cells. Moreover, the combination achieved greater anti-tumor effects on mesothelioma developed in the pleural cavity than administration of either ZOL or CDDP alone. ZOL-treated cells as well as CDDP-treated cells induced p53 phosphorylation at Ser 15, a marker of p53 activation, and up-regulated p53 protein expression levels. Down-regulation of p53 levels with siRNA however did not influence the ZOL-mediated cytotoxicity but negated the combinatory effects by ZOL and CDDP. In addition, ZOL treatments augmented cytotoxicity of adenoviruses expressing the p53 gene on mesothelioma. These data demonstrated that ZOL-mediated augmentation of p53, which was not linked with ZOL-induced cytotoxicity, played a role in the combinatory effects with a p53 up-regulating agent, and suggests a possible clinical use of ZOL to mesothelioma with anti-cancer agents.     

Studies on mutagenesis and repair induced by platinum analogs

Mutagenesis and cytotoxicity were studied in Escherichia coli by iproplatin and carboplatin, two analogs of cisplatin (CDDP) currently undergoing clinical trial. As with CDDP, mutagenesis by these agents was mediated by the umuDC gene product. In contrast to CDDP, however, mismatch repair did not substantially contribute to survival of cells after exposure to these agents since dam-3 E. coli were not more sensitive than wild type E. coli. UvrA- E. coli, however were more sensitive to these analogs demonstrating that as with CDDP, uvr endonuclease-mediated excision contributes to the repair of DNA damage induced by platinum compounds.     

Chemotherapy drug delivery from calcium phosphate nanoparticles

Calcium phosphate nanoparticles (nanoCaP) conjugated with cis-diamminedichloroplatinum (CDDP, cisplatin) were prepared through the electrostatic binding of an aquated species of cisplatin to the nanoCaP in a chloride-free solution. The agglomeration of the nanoCaP that typically occurs during synthesis of CaP was controlled through the addition of DARVAN 811 immediately after precipitation and before drug conjugation. In vitro drug release studies were completed and showed a sustained release of CDDP from the nanoconjugates over time. The cytotoxicity of the nanoCaP/CDDP was compared to that of the free drug in an in vitro cell proliferation assay using the CDDP resistant A2780cis human ovarian cancer cell line. The CDDP released from the nanoconjugates was equally effective as the free drug against the A2780cis cell line. Direct addition cytotoxicity studies revealed that the sterically-stabilized, negatively-charged drug nanoconjugates are unable to overcome drug resistance and had an increased IC50 value as compared to the free drug.     

Impact of intracellular chloride concentration on cisplatin accumulation in sensitive and resistant GLC4 cells

Resistance to cisplatin [cis-diamminedichloroplatinum(II), CDDP] chemotherapy is a major problem in the clinic. Understanding the molecular basis of the intracellular accumulation of CDDP and other platinum-based anticancer drugs is of importance in delineating the mechanism of resistance to these clinically important therapies. Different molecular mechanisms may coexist, but defective uptake of CDDP is one of the most consistently identified characteristics of cells selected for CDDP resistance. We have studied the impact of intracellular chloride concentration on platinum-based compound accumulation in the human GLC4, GLC4/CDDP, and K562 tumor cell lines. We show that (1) a decrease of intracellular chloride concentration yielded an increase of CDDP accumulation and vice versa and (2) the intracellular chloride concentration in GLC4/CDDP cells is higher than in sensitive cells, whereas CDDP accumulation shows the opposite behavior. The identification of chloride as a critical determinant of CDDP intracellular accumulation and the molecular mechanisms by which CDDP-resistant cells modulate chloride concentration may allow alternative therapeutic approaches. Our findings indicate that increase of intracellular chloride concentration may be a major determinant of CDDP resistance.     

Synergistic interaction between cisplatin and PARP inhibitors in non-small cell lung cancer

The antineoplastic agent cis-diammineplatinum(II) dichloride (cisplatin, CDDP) is part of the poorly effective standard treatment of non-small cell lung carcinoma (NSCLC). Here, we report a novel strategy to improve the efficacy of CDDP. In conditions in which CDDP alone or either of two PARP inhibitors, PJ34 hydrochloride hydrate or CEP 8983, used as standalone treatments were inefficient in killing NSCLC cells, the combination of CDDP plus PJ34 or that of CDDP plus CEP 8983 were found to kill a substantial fraction of the cells. This cytotoxic synergy could be recapitulated by combining CDDP and the siRNA-mediated depletion of the principal PARP isoform, PARP1, indicating that it is mediated by on-target effects of PJ34 or CEP 8983. CDDP and PARP inhibitors synergized in inducing DNA damage foci, mitochondrial membrane permeabilization leading to cytochrome c release, and dissipation of the inner transmembrane potential, caspase activation, plasma membrane rupture and loss of clonogenic potential in NSCLC cells. Collectively, our results indicate that CDDP can be advantageously combined with PARP inhibitors to kill several NSCLC cell lines, independently from their p53 status. Combined treatment with CDDP and PARP inhibitors elicits the intrinsic pathway of apoptosis.     

Cytokinetic effects of carboplatin and cisplatin on a human ovarian cancer cell line

The cytokinetic effects of carboplatin(CBDCA) on a human ovarian cancer cell line(KF-1) were examined by means of cell survival rate and flow cytometry in comparison with cisplatin(CDDP). CBDCA and CDDP exhibited dose dependent cytotoxicity on KF-1, and CBDCA showed compatible cell growth inhibition to that of 15 times concentration of CDDP in comparison with IC50 of 72 hrs after drug addition. From the analysis of cell cycle, CBDCA and CDDP inhibited cell cycle progression at G2 + M phase. CBDCA exhibited G2 + M phase block to that of 15 to 20 times the concentration of CDDP. We suggested that CBDCA had potential therapeutic activity against ovarian cancer, but should be evaluated carefully in the clinical use.     

Differential gene expression profile between PC-14 cells treated with free cisplatin and cisplatin-incorporated polymeric micelles

Cisplatin (CDDP)-incorporated polymeric micelles (CDDP/m) are a macromolecular carrier system possessing a time-modulated decaying property accompanied by sustained release of free drug. The gene expression profiles in nonsmall cell lung cancer PC-14 cells treated with free CDDP and CDDP/m were evaluated by a cDNA expression array for 807 genes. Although the total gene expression profile of the cells treated with CDDP/m approximated that of free CDDP in the hierarchical clustering analysis, a number of genes showed differential expression according to whether the cells had been treated with CDDP or CDDP/m. Ultimately, 50 genes with significant differential expression between cells treated with CDDP and CDDP/m were selected by principal component (PC) analysis and the unpaired t-test. The genes selected, including genes related to cell cycle regulation, apoptosis-related proteins, detoxification, and DNA repair enzymes, were considered to be related to CDDP-induced cytotoxicity. Interestingly, CDDP/m down-regulated the genes encoding integrins and matrix metalloproteinases (MMPs), which play an integral role in tumor invasion, metastasis, and angiogenesis, whereas free CDDP up-regulated them. The results suggest that use of the macromolecular carriers may yield additional therapeutic effects over free drug.     

In vitro inhibitory activity of EDTA against planktonic and adherent cells ofCandida sp.

Candida sp. can cause infections of indwelling medical devices associated with biofilm formation, which are difficult to treat due to insensitivity of adherent microorganisms to host defence mechanisms and standard antimicrobial therapy. The aim of this paper was to determine the effect of EDTA (disodium salt) on the adhesion ofCandida sp. to some catheters and also on biofilm formation by the yeasts and its eradication in relation to cytotoxicity of this chelating agent to the cell cultures. The adhesion process and biofilm formation, and also EDTA cytotoxicity to green monkey kidney (GMK) cell culture were determined using MTT tetrazolium salt [3-(4,5-dimethylthiazol-2-yl) ?2,5-diphenyltetrazolium bromide)] reduction assay. EDTA inhibited the growth of free-floating forms ofCandida sp. strains with minimal inhibitory concentration (MIC) from 0.06 to 0.25 mM; the minimal fungicidal concentration (MFC) values ranged from 64 to 128 mM. The prevention ofCandida sp. adhesion on the catheters used or eradication of the adherent cells was achieved at 0.5 to 4.0 mM EDTA. Also biofilm formation was prevented by 0.5 to 4.0 mM EDTA. Much higher concentration of EDTA (32 to 128 mM) was needed to eradicate the mature biofilm. EDTA at concentration up to 1 mM was not toxic for GMK cells. At higher concentration, toxicity of EDTA to GMK cells was correlated with the concentration of this agent and the time of exposure. Summing up, EDTA may be regarded as a useful agent rather in prophylaxis of candidal infections of medical devices.     

Intracellular uptake of an antitumor-active azole-bridged dinuclear platinum(II) complex in cisplatin-resistant tumor cells

A cationic azolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH₃)₂}₂(μ-OH)(μ-methyl-pyrazolate)]²⁺ (4M-PzPt), was developed to overcome resistance to cisplatin (CDDP). This study aimed to assess the cytotoxicity of 4M-PzPt against a CDDP-resistant cell line, H4-II-E/CDDP, and compare the intracellular accumulation of CDDP and 4M-PzPt. H4-II-E and H4-II-E/CDDP displayed similar sensitivity to 4M-PzPt; however, the sensitivity of H4-II-E/CDDP to CDDP was approximately 19-fold lower than that of H4-II-E. The difference in the sensitivity to both platinum complexes corresponded with the difference in the amount of intracellular platinum accumulation after exposure to CDDP or 4M-PzPt in both cell lines. In H4-II-E, HepG2, and HuH-7 cells, the intracellular uptake of CDDP and 4M-PzPt occurred via active transport and passive transport. Results of co-exposure with the transport inhibitors ouabain, tetraethylammonium, and cimetidine indicated that the intracellular uptake of CDDP was dependent on Na⁺/K⁺-ATPase and that of 4M-PzPt was dependent on organic cation transporters (OCTs), probably OCT1. This study suggested that 4M-PzPt could inhibit the growth of a CDDP-resistant tumor via an intracellular uptake mechanism different from that of CDDP.     

Evaluations of Anticancer Effects of Combinations of Cisplatin and Tirucallane-Type Triterpenes Isolated from Amphipterygium adstringens (Schltdl).

The cytotoxic activity of combinations of masticadienonic (AMD) or 3αOH-hydroxy-masticadienonic (3αOH-AMD) acids with cisplatin (CDDP) was evaluated against PC3 prostate and HCT116 colon cancer cell lines. Combinations A (half the IC₅₀ value), B (IC₅₀ value), and C (twice the IC₅₀ value) were tested at a 1 : 1 ratio. All AMD plus CDDP combinations demonstrated increased cytotoxic effect, as determined by the sulforhodamine B test, in both cell types. The best combination was B, which showed 93 % and 91 % inhibition of the proliferation of PC3 and HCT116 cells, respectively. It also increased apoptosis in the PC3 cell lines, as evaluated by flow cytometry. However, in vivo tests showed no additional activity from the AMD plus CDDP combinations. These results showed that the increased cytotoxic activity of the combinations in vitro did not reflect in vivo tests. All combinations of 3αOH-AMD plus CDDP exerted antagonistic effects in both cell types.     

Toxicity of cisplatin to the central nervous system of male rabbits

The cytotoxic effects of platinum (Pt) were studied by intraparenchymal injection of 1 mg of cisplatin (CDDP) in male rabbits. Time-serial plasma Pt levels were used as CDDP clearance indices in brain and kidney tissues. The tissue samples were also examined histologically. Changes in the blood-brain barrier (BBB) were evaluated by horseradish peroxidase (HRP) extravasation. In the brain infusion group, Pt was detected in the plasma 30 min after the start of infusion. In the kidney, Pt was detected after 10 min of CDDP injection. The maximum plasma concentration of Pt in the brain group showed diffuse edema, neuronal necrosis, karyolysis, and HRP extravasation around the injection site. In contrast, the histological damage to kidneys was minimal. The results presented here show that direct infusion of CDDP caused the most extensive cytotoxicity in the brain. The low clearance rate of CDDP from the brain and BBB disruption may explain this behavior.     

Pharmacological Modulation of Cisplatin Toxicity Rhythms with Buthionine Sulfoximine in Mice Bearing Pancreatic Adenocarcinoma (PO3)

In a previous report, we showed that the circadian rhythm of cisplatin (cis-diamminedichloroplatinum, CDDP) toxicity in healthy mice was modified by buthionine sulfoximine (BSO), a specific inhibitor of glutathione (GSH) synthesis. In the present study, the effects of BSO on the rhythms of CDDP toxicity and antitumor efficacy were investigated in mice bearing a transplantable pancreatic adenocarcinoma (PO3). B₆D₂F₁ mice were inoculated widi two 4 mm³ tumor fragments, one in each flank, then were synchronized with an alternation of 12h of light (L) and 12h of darkness (D) (LD 12: 12). Three weeks later, a single dose of CDDP (12 mg/kg iv) was injected at 3h, 7h, 11h, 15h, 19h, or 23h after light onset (HALO) with or without prior BSO (450 mg/kg ip 4h earlier). The antitumor activity of CDDP as assessed by tumor weight change and tumor growth delay was weak in this tumor model irrespective of prior BSO administration or CDDP dosing time. Nevertheless, toxic effects of CDDP as gauged by body weight loss or survival varied significantly according to CDDP dosing time. Body weight loss was least in mice receiving CDDP alone at the mid-to-late active span. Survival rate was 97% in mice treated with CDDP alone and 47% in those receiving prior BSO (χ² = 23.6, p <. 0001). BSO pretreatment further shifted the period of survival or body weight change from 24h to (10 + 24)h, an effect similar to that earlier reported in healthy mice. Thus, PO3 tumor at a measurable stage altered neither the circadian rhythm in CDDP toxicity nor the ultradian rhythm in the toxicity of BSO-CDDP combination. The results suggest that rhythms in target tissues for drug actions can be manipulated with biochemical modulators, thus partly escaping central clock control.