1,3 Bis (2-chloroethyl)-1-nitrosourea-induced host resistance to syngeneic LSA mouse leukemia

A syngeneic tumor, the LSA lymphoma of C57BL mice, is considered non-antigenic or only weakly antigenic because no host reaction was elicited after repeated exposure of mice to killed tumor. However, easily detectable complete tumor resistance was elicited after 1 or 2 exposures to BCNU and tumor cure. Marked amplification of this host immunity in the BCNU treatment-cured mouse was shown by serial graded exposures to viable tumor grafts, and it was possible to establish virtually complete resistance to further tumor challenge by this method. A lymphoma spleen colony forming assay detected the antigenic properties of the LSA lymphoma in syngeneic C57BL mice. The host resistance state was easily prevented by sublethal irradiation of the mouse with 300 to 400 rad before tumor implantation. Once resistance was established, it was not altered by 450 rad of total body irradiation. These differences indicate a radiosensitive cell for the adaptive immune response and a less radiosensitive (and more complex) mechanism for the established tumor resistant state.     

Comparative analysis of temozolomide (TMZ) versus 1,3-bis (2-chloroethyl)-1 nitrosourea (BCNU) in newly diagnosed glioblastoma multiforme (GBM) patients

PURPOSE: Although TMZ replaced BCNU as the standard initial chemotherapy in the treatment of GBM, no studies have been reported comparing BCNU with TMZ. We therefore did a retrospective analysis comparing these agents as initial therapy in GBM. PATIENTS AND METHODS: Eighty-one patients with GBM in our institution received both radiation and chemotherapy as initial treatment after surgery or biopsy; 49 receiving BCNU and 32 TMZ. These were analyzed for overall survival (OS) and progression-free survival (PFS) versus the type of chemotherapy used. The influence of salvage therapy on the outcome was investigated also. RESULTS: Median OS was superior in the TMZ versus the BCNU group (15.9 vs. 11.5 months) and the curves were judged to be significantly different by the log-rank test; P < 0.02. However, PFS was not significantly different between the two groups. Bevacizumab plus irinotecan (BI) was used as salvage therapy in one-third of the TMZ patients but in none of the BCNU patients. When patients receiving BI were omitted from the TMZ group the OS curve overlapped that of BCNU patients. CONCLUSION: These data suggest that the superior OS of the TMZ-treated GBM patients was not due to better tumor control by TMZ but was possibly related to the newer salvage therapy that was available to them.     

Specific mTOR inhibitor rapamycin enhances cytotoxicity induced by alkylating agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) in human U251 malignant glioma cells

Loss of the PTEN tumor suppressor gene and amplification of the epidermal growth factor receptor (EGFR), which is common in malignant gliomas, result in activation of the mammalian target of rapamycin (mTOR). Rapamycin is a highly specific inhibitor of mTOR and induces a cytostatic effect in various glioma cell lines. DNA-damaging agents such as nitrosourea are widely used in malignant glioma treatment; therefore, we investigated the effect of rapamycin on cell growth and death in combination with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU, nimustine hydrochloride) in human glioma cells. In U251 malignant glioma (U251MG) cells, we confirmed that rapamycin enhanced ACNU-induced apoptosis. We found that rapamysin inhibited ACNU-induced p21 induction, and knocking down of p21 protein by siRNA enhanced ACNU-induced apoptosis in U251MG cells. Furthermore, adenovirus-mediated over-expression of p21 protein rescued U251MG cells from apoptosis induced by ACNU and rapamycin. Finally, treatment of intracerebral U251MG xenografts with a combination of rapamycin and ACNU in vivo resulted in statistically prolonged median survival (P < 0.05). These results suggest that rapamycin in combination with DNA-damaging agents may be efficacious in the treatment of malignant gliomas.     

Failure of adjuvant immunotherapy to prevent central nervous system metastases in malignant melanoma patients.

A review of our experience using BCG immunotherapy as a postsurgical adjunct in the treatment of melanoma shows that the incidence of systemic metastases appears to have been reduced. However, central nervous system (CNS) metastases continue to develop in these patients and represent the single most frequent cause of death. Serial studies of immune competence in these patients reveal that those with CNS metastases usually retain normal immune responses, whereas those with metastases at other sites exhibit progressive immunosuppression with advancing disease.     

1,1-双(4-氯苯)-2,2,2-三氯乙烷对大鼠神经系统的氧化应激作用

背景与目的：研究1,1-双(4-氯苯)-2,2,2-三氯乙烷（DDT）对大鼠大脑皮层、海马和小脑组织诱导的氧化应激作用。 材料与方法：SD大鼠共24只, 随机分为4组, 即溶剂对照组与低 （20mg/kg）、中 （40mg/kg）、高（80mg/kg）3个DDT剂量组,每组6只,大鼠经口灌胃染毒7 d后断头处死,测定大脑皮层、海马和小脑组织丙二醛（MDA）水平和总超氧化物歧化酶（T-SOD）、谷胱甘肽过氧化物酶（GSH-PX）和谷胱甘肽还原酶(GR)活力。 结果：①与溶剂对照组比较,随着DDT浓度的增加,大鼠大脑皮层、海马和小脑中MDA含量升高,T-SOD活性下降;② 大脑皮层GSH-PX活性在低剂量时升高,在中、高剂量组则显著下降,海马GSH-PX活性随着剂量的增加而显著下降,而小脑GSH-PX活性则在中、高剂量组出现显著下降;③ 海马GR活性在中、高剂量组随着剂量的增加而显著下降,在小脑则只有高剂量组出现了下降,皮层GR活性在各剂量组未观察到明显改变。结论：DDT可以引发神经组织的脂质过氧化反应增强。DDT导致机体组织的氧化损伤可能在动物神经毒性中起重要作用。     

Neurotoxicity and pharmacokinetics of ventriculolumbar perfusion of methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-gluco-pyranoside (MCNU) in dogs

Summary Ventriculolumbar perfusion of methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-glucopyranoside (MCNU), a water soluble nitrosourea with log P-0.71, may be efficacious in the treatment of subarachnoid dissemination of malignant glioma. We used 2 dogs to study the neurotoxicity and pharmacokinetics of MCNU. MCNU (1 mg), dissolved in 10 ml of artificial CSF, was administered via the right lateral ventricle during a period of 18 to 42 min and the CSF was drained by lumbar puncture. The perfusion was repeated once a week for 10 consecutive weeks. No neurological and systemic symptoms were noted after perfusion. Histological examination of the brain and spinal cord showed local denudation of the ependyma and local subependymal spongy degeneration and gliosis in the lateral ventricle into which MCNU was administered in one dog and local denudation of the ependyma in the other. When administration was over a period of 21 to 38 min, the MCNU concentration in the lumbar CSF peaked at 11.11 to 50.67 g/ml, in 28 to 78 min. The area under the drug concentration-time curve (AUC) was 1152 g×min/ml on average, significantly larger than that of ACNU. The elimination phase followed linear kinetics and the half-time was 41.1 min on average, significantly longer than that of ACNU. These findings suggest that ventriculolumbar perfusion of MCNU may be effective in the treatment of subarachnoid dissemination of malignant glioma notwithstanding some local histological changes.     

Sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea of the EMT6 tumor in vivo as determined by both tumor volume response and in vitro plating assay

Measurements of the response of the EMT6 mouse tumor to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea by either tumor volume or the in vitro assay of the cell surviving fraction give very different results. For BCNU very little delay in tumor growth is caused by high doses of the drug, whereas the surviving fraction assayed 2 hr after drug administration may be as low as 10(-4) for comparable drug doses. Over the first 48 hr after BCNU, the measured surviving fraction in small tumors increases 50- to 100-fold; this is ascribed to the phenomenon known as "recovery from potentially lethal damage." For 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, however, this early rapid recovery in surviving fraction does not occur. Although the surviving fractions measured 24 hr after drug administration are similar for equivalent doses of the two drugs, the growth delay induced is very much longer for 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea than for BCNU. Neither of the agents appears to cause any increase in the rate of cell loss from tumors as measured by [125]iododeoxyuridine.     

Effects of K-ras Gene Mutations in the Development of Lung Lesions Induced by 4-(N- Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone in A/J Mice

The relationship between the development of peripheral lung lesions induced by tobacco-specific 4-( N -methyl- N -nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and K- ras gene mutation in A/J mice, and the correlations between histological alterations and the course of lung lesion development after NNK treatment and K- ras gene mutation were investigated. The acquisition of a selective growth advantage by the lung lesions with mutations was also examined using immunohistochemical labeling with bromodeoxyuridine. Thirty female 5 weeks old A/J mice were each injected intraperitoneally with a single dose of NNK (100 mg/kg body weight) and subdivided into 6 groups according to the time after NNK treatment. The lung lesions were characterized histologically as alveolar/bronchiolar hyperplasia, adenoma and adenocarcinoma, and point mutations in codons 12 and 61 of the K- ras gene were detected by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and dideoxy sequencing methods. K- ras gene mutations were identified in 7 (58.3%) of 12 hyperplasias, 42 (75.0%) of 56 adenomas and 3 (75.0%) of 4 adenocarcinomas. The most frequent K- ras gene mutation was a G-to-A transition at the second base of codon 12 and this accounted for 86.5% of all the mutations detected. Neither the frequency of activation of this gene nor the specific mutation was affected by the time after NNK treatment and there was no positive correlation between the proliferative activity of lung lesions and the presence of K- ras gene mutations. Thus, K- ras gene mutation is closely associated with the development of NNK-induced peripheral lung lesions in A/J mice, but it plays no role in the selective growth advantage of these lesions.     

Contribution of O6-methylguanine-DNA methyltransferase to resistance to 1,3-(2-chloroethyl)-1-nitrosourea in human brain tumor-derived cell lines

To assess the possible role of the DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) in resistance of brain neoplasms to the clinically important chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), we quantitated MGMT activity, BCNU survival, and the effect of ablating MGMT activity on the sensitivity of 14 human medulloblastoma- and glioma-derived cell lines. BCNU resistance, measured as 10% survival dose (LD10), differed eightfold among the lines. Elimination of measurable MGMT activity with the substrate analogue inhibitor O⁶-benzylguanine (O⁶-BG) revealed a variable but limited contribution of MGMT to survival. In no case did O⁶-BG reduce LD₁₀ by more than 3.4-fold. In contrast, 06-BG reduced the LD10 for N-methyl-N′-nitro-N-nitrosoguanidine up to 31-fold in the same cell lines (Bobola MS, Blank A, Berger MS, Silber JR, Mol Carcinog 13:70–80, 1995). Variability in BCNU survival, manifested as a sevenfold range of LD₁₀, persists after measurable MGMT was eliminated, indicating that another mechanism or mechanisms is operating to limit cytotoxicity. Cells alkylated while suspended in growth medium are more resistant to BCNU and display less dependence on MGMT than cells treated while proliferating on a plastic substratum. When alkylated in suspension, most of the lines are either unresponsive to O⁶-BG or contain a subpopulation that did not respond to O6-BG. Our results demonstrate that BCNU resistance is multifactorial and that MGMT makes a modest contribution to resistance in our lines. © 1995 Wiley-Liss, Inc.     

The sensitivity of cells in exponential and stationary phases of growth to bleomycin and to 1,3-bis(2-chloroethyl)-1-nitrosourea

Studies of EMT6 mouse tumour cells growing in vitro have shown that these cells become less sensitive to bleomycin as they pass from exponential growth into stationary phase. This result is the opposite of that recently reported by two other groups of workers using different cell systems, but in agreement with the results of a third group of workers. Results for 1,3-bis(2-chloroethyl)-1-nitrosourea, however, confirm the findings of other workers that cells become more sensitive to this agent as they pass into stationary phase.     

Intraarterial O6-benzylguanine enables the specific therapy of nitrosourea-resistant intracranial human glioma xenografts in athymic rats with 1,3-bis(2-chloroethyl)-1-nitrosourea

 The prognosis for patients with malignant gliomas continues to be dismal. The high degree of resistance of gliomas to nitrosourea-based chemotherapy is one major factor in poor treatment outcome. The identification of O ⁶-alkylguanine-DNA alkyltransferase (AGAT) as a major determinant of nitrosourea resistance has resulted in the development of several agents to inactivate this repair protein and counteract tumor cell resistance. However, a major problem in preclinical trials has been the marked nitrosourea dose limitations imposed by the prior administration of AGAT-depleting agents. We investigated the AGAT depletion and selective enhancement of BCNU activity of intraarterial (i.a.) O ⁶-benzylguanine (O ⁶BG) in the human malignant glioma xenograft D-456 MG growing intracranially (i.c.) in athymic rats. Whereas i.a. O ⁶BG at 2.5 mg/kg produced 100% inhibition of D-456 MG AGAT i.c. activity 8 h after administration, intraperitoneal (i.p.) O ⁶BG at this dose produced only 40% inhibition, requiring dose escalation to 10 mg/kg to produce 100% AGAT depletion. Prior administration of i.p. O ⁶BG (10 mg/kg) and i.a. O ⁶BG (2.5 mg/kg) limited maximum tolerated intravenous (i.v.) BCNU doses (37.5 mg/kg when given alone) to 6.25 and 25 mg/kg, respectively. Higher doses of BCNU alone or in combination with O ⁶BG produced histopathologic evidence of cerebral and hepatic toxicity. Therapy experiments revealed a significantly improved median survival for rats treated with O ⁶BG i.a. (2.5 mg/kg) plus BCNU i.v. (25 mg/kg, days 61 and 59 in duplicate experiments) compared with saline (day 21, P=0.001), O ⁶BG i.a. or i.p. (days 22 and 23, P=0.001), BCNU i.v. (37.5 mg/kg, day 29, P=0.001), and O ⁶BG i.p. (10 mg/kg) plus BCNU i.v. (6.25 mg/kg, day 37, P<0.001). Therefore, O ⁶BG i.a., by virtue of rapid AGAT depletion and selective uptake into i.c. tumors, offers significant potential for regional chemomodulation of AGAT-mediated nitrosourea resistance in malignant human gliomas with concomitant reduction of systemic toxicity. Received: 30 January 1996/Accepted: 16 June 1996     

Local Delivery of Minocycline and Systemic BCNU have Synergistic Activity in the Treatment of Intracranial Glioma

Minocycline, a tetracycline derivative, has been shown to inhibit tumor angiogenesis through inhibitory effects on matrix metalloproteinases. Previous studies have shown this agent to be effective against a rodent brain tumor model when delivered intracranially and to potentiate the efficacy of standard chemotherapeutic agents. In the present study, the in vivo efficacy of intracranial minocycline delivered by a biodegradable controlled-release polymer against rat intracranial 9L gliosarcoma was investigated to determine whether it potentiates the effects of systemic 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Minocycline was incorporated into the biodegradable polymer polyanhydride poly[bis(p-carboxyphenoxy)propane-sebacic acid] (pCPP:SA) at a ratio of 50:50 by weight. The release kinetics of minocycline from the polymer were assessed. For the efficacy studies, female Fischer 344 rats were implanted with 9L glioma. Treatment with minocycline delivered by the pCPP:SA polymer at the time of tumor implantation resulted in 100% survival in contrast to untreated control animals that died within 21 days. Treatment with the minocycline-polymer 5 days after tumor implantation provided only modest increases in survival. The combination of intracranial minocycline and systemic BCNU extended median survival by 82% compared to BCNU alone (p < 0.0001) and 200% compared to no treatment (p < 0.004). We conclude that local intracranial delivery of minocycline from biodegradable controlled-release polymers inhibits tumor growth and may have clinical utility when combined with a chemotherapeutic agent.     

Mode of Action of Estra-1,3,5(10)-triene-3,17β-diol 3-Benzoate 17-((4-(4-Bis(2-chloroethyl)amino)phenyl)-1-oxobutoxy)acetate) on Human Breast Carcinoma Xenografts in Nude Mice

To elucidate the mode of action of busramustine (KM2210), 17β- and α-busramustine, estradiol and chlorambucil were used for experimental chemo- and endocrino-therapy against hormone-dependent (T-61) and independent (MX-1) human breast carcinomas serially transplanted into BALB/cA female nude mice. Busramustine was administered po daily for 3 weeks at doses of 12.5–300 mg/kg for the β-isomer and 25–300 mg/kg for the α-isomer. Five to 50 mg of estradiol per kg was administered im once, and 3 to 6 mg of chlorambucil per kg was administered po daily for 3 weeks. All of the compounds were effective against estrogen receptor-positive T-61 with a clear dose-response relationship, while estrogen receptor-negative MX-1 was sensitive to all of the agents except estradiol. Since the α-isomer of busramustine was effective against both tumor lines, the mode of action of 17β-busramustine may not be related to estrogenic action by estradiol released from the maternal compound. However, 17bT-busramustine generated the estrogen receptor system of T-61 tumor and resulted in the endometrial hyperplasia of tumor-bearing nude mice, suggesting that this compound also has estrogenic action on transplanted human breast carcinoma and tumor-bearing host mice, besides non-estrogenic antitumor activity on human breast carcinoma xenografts.     

Inhibition of O6-alkylguanine-DNA-alkyltransferase activity potentiates cytotoxicity and induction of SCEs in human glioma cells resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea

SF-188 is a human glioma-derived cell line resistant to thecytotoxic effects of and the induction of sister chromatid exchanges(SCEs) by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Pretreatmentof SF-188 cells with N-methyl-N-nitrosourea (MNU) for 1 h increasedthe cytotoxicity of a 1-h treatment with BCNU 2-to 10-fold anddoubled the number of SCEs; the magnitude of these effects wasdependent on the dose of both agents. Treatment of SF-188 cellswith MNU resulted in a dose-dependent inhibition of O⁶-alkylguanine-DNA-alkyltransferase(O⁶-AT) activity. Low doses of MNU, which did not significantlyinhibit O⁶-AT, did not potentiate SCE induction. Higher dosesof MNU inhibited O⁶-AT and potentiated cytotoxicity and theinduction of SCEs. These results are consistent with the hypothesisthat in resistant cells treated with BCNU, O⁶-AT repairs O⁶-chloroethylguaninebefore it can form a DNA interstrand cross-link. Inhibitionof this enzyme allows for the formation of BCNU-induced DNAinterstrand cross-links resulting in increases in cytotoxicityand induction of SCEs. The correlation between cytotoxicityand the induction of SCEs suggests that measurement of SCEsmay be useful for determining the cellular response of normaland tumor cells to in vivo treatment with combinations of chemotherapeuticagents.     

2-（3-羧基-1-丙酰氨基）-2-脱氧-D-葡萄糖对人食管癌细胞Eta-109的致凋亡研究

目的 探讨2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖是否具有诱导人食管癌细胞Eta-109发生凋亡的作用。方法 采用体外细胞培养实验方法，应用MTT法、流式细胞仪检测、DNA琼脂糖凝胶电泳、透射电镜等技术，观察药物对细胞生长的抑制率及细胞的形态学变化，检测凋亡峰及DNALadder。结果 MTT法测得细胞抑制率具有显著的时间及浓度依赖性；透射电镜下可见到凋亡典型的形态学变化，流式细胞仪检测显示细胞周期的G期阻滞及凋亡峰；DNA琼脂糖凝胶电泳显示清晰的DNA梯状电泳。结论 2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖能够明显的抑制人食管癌细胞Eca-109的增殖，并诱导其发生凋亡。     

2-(α-羟基戊基)苯甲酸钾盐的急性毒性和致突变作用研究

目的:研究2-(α-羟基戊基)苯甲酸钾盐(dl-PHPB)的急性毒性及致突变性。方法:急性毒性试验采用Bliss法,昆明小鼠分为5组,每组10只,雌雄各半,分别按256.0、286.3、320.0、357.8、400.0、447.2 mg/kg静脉注射dl-PHPB,观察注射后至给药后14 d动物的毒性反应性质及程度,计算半数致死剂量(LD₅₀)及95%可信限;Ames试验采用平板掺入预培养法,选用鼠伤寒沙门氏菌株TA97、TA98、TA100和TA102,每皿0.5～5 000.0 μg浓度范围内,检测加和不加S9条件下对Ames菌的致突变性;微核试验采用雄性昆明小鼠,分为3组,每组6只,分别按23.3、70、210 mg/kg单次静脉注射dl-PHPB,注射后24 h计数骨髓嗜多染红细胞微核率(MNPCEs);体外染色体畸变试验在加和不加S9条件下,检测11.0~88.0 μg/mL dl-PHPB对CHL细胞染色体畸变率的影响。结果:小鼠静脉注射dl-PHPB后,各剂量动物出现一过性呼吸急促、自发活动减少、俯卧少动等症状,随剂量增加至≥320.0 mg/kg时,部分动物出现濒死症状,并在给药后2~30 min内死亡,死亡率随剂量的增加而增加,256.0~447.2 mg/kg 6个剂量组死亡率依次为0、0、10%、30%、70%、100%。未死亡动物在给药30 min后逐渐恢复正常。14 d观察期期间动物未见其他异常。其LD₅₀为373.3 mg/kg,95%可信限为355.6～392.0 mg/kg;Ames试验结果显示,在每皿0.5～5 000.0 μg浓度范围内未引起4种测试菌株回复突变数增加;微核试验结果显示,dl-PHPB在23.3~210.0 mg/kg范围内,各组动物MNPCEs均低于4‰;CHL细胞体外染色体畸变试验中,dl-PHPB在 11.0～88.0 μg/mL浓度范围内致CHL细胞染色体畸变率<5%。结论:在本实验条件下,小鼠静脉注射dl-PHPB的LD₅₀为373.3 mg/kg,致突变性3项试验均为阴性结果。     

The use of immunotherapy to enhance tumor rejection immunity in leukemic (L2C) guinea pigs following remission induction with MeCCNU

Employing a transplantable guinea pig leukemia (L₂C), animals treated with MeCCNU were completely free of disease 180 days post chemotherapy. However, these animals were not refractory to a subsequent challenge with viable L₂C leukemic blast cells. In an attempt to augment the host's immune response to tumor associated antigens (TAA) while enhancing the immunogenicity and expression of the TAA on the surface of the blast cell, guinea pigs were injected with either irradiated syngeneic blast cells, immunostimulators (BCG or C. parvum) or combinations of both. Guinea pigs treated with either BCG or C. parvum plus irradiated cells were now resistant to tumor re-inoculation. Use of irradiated blast cells or BCG or C. parvum alone was ineffective and did not protect against a bio-challenge. Therapeutic benefit was only observed following the mixture of vaccine with the immunostimulator. Application of immunostimulation at sites separate from the vaccine was less effective. Maximum stimulation of the immune response in the form of tumor rejection immunity occurred following repeated immunization (3 times vs 1 time) every other week with the vaccine-immunostimulator mixture. These results were substantiated by in vitro studies in which significant increases in both cellular and humoral mediated immunity were noted in only those animals receiving the combined mixture of irradiated blasts plus immunostimulator. These data suggest the need for enhancing tumor-specific recognition in situations where the host is immunologically competent but the TAA are too weak to elicit an immune response beneficial to the host.     

Antifungal and cancer cell growth inhibitory activities of 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene

The antifungal and cancer cell growth inhibitory activities of 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene (TMPN) were examined. TMPN was fungicidal for the majority of 132 reference strains and clinical isolates tested, including those resistant to fluconazole, ketoconazole, amphotericin B or flucytosine. Minimum fungicidal concentration/minimum inhibitory concentration (MFC/MIC) ratios were < or = 2 for 96% of Cryptococcus neoformans clinical isolates and 71% of Candida albicans clinical isolates. TMPN was fungicidal for a variety of other basidiomycetes, endomycetes and hyphomycetes, and its activity was unaffected by alterations in media pH. The frequency of occurrence of fungal spontaneous mutations to resistance was <10(-6). Kill-curve analyses confirmed the fungicidal action of TMPN, and demonstrated that killing was concentration- and time-dependent. At sub-MIC exposure to TMPN, C. albicans did not exhibit yeast/hyphae switching. TMPN was slightly cytotoxic for murine and human cancer cell lines (GI50=1-4 microg ml(-1)), and weakly inhibited mammalian tubulin polymerization (IC50=0.60 microg ml(-1)).