An indoline-derived compound that markedly reduces mouse body weight.

OBJECTIVE: We describe how a single intraperitoneal injection of an indoline-derived drug (SN 28127) reduced mouse body weight (25-45% loss) and adipose tissue mass (∼75%). METHODS AND RESULTS: The reductions in body weight peaked at ∼21-28 days post drug injection and were maintained throughout the study (160 days). The mice ate as much as vehicle-treated control mice. A more potent SN 28127 analog (SN 29220) reversed high-fat diet-induced obesity and type 2 diabetes in C57BL/6J mice on a high-fat diet. Insulin induced a sustained reduction in blood glucose in fasted SN 29220-treated mice compared with the vehicle-treated mice. All drug-treated mice exhibited a transient increase in water intake from ∼10 days post drug injection that lasted for ∼70 days. Following a single injection of (3)H-labeled SN 29220, radioactivity accumulated within 4 h in the liver, bile duct and ileum with little detected in the brain; within 1-2 days, most of the radioactivity was found in the pancreas, spleen, liver, bile duct, stomach, kidneys and white adipose tissue. High levels of glucose were detected in urine collected from SN 29220 but not vehicle-treated C57BL/6J mice at ∼60 days post injection, while fecal triacylglycerols and cholesterol were not different between SN 29220 and vehicle-treated mice. These data lead us to hypothesize that the hepatic system is the primary drug target. Genes involved in fatty acid synthesis (FASn, SCD1 and PPARγ) and appetite stimulation (AGRP) were upregulated at 160 days post drug treatment, indicative of adaptation to reduced body weight. CONCLUSION: We hypothesize that indoline-derived drug-induced chronic toxicity to the hepatic system leads to a reduction in white adipose tissue mass. The mice adapt to this drug-induced toxicity with reduced steady-state body weight. Understanding molecular mechanisms underlying these responses has potential to identify novel targets for prevention and treatment of obesity.

Efficacy and safety of rifampin containing regimen for staphylococcal prosthetic joint infections treated with debridement and retention.

The aim of this study was to evaluate the efficacy and safety of rifampin for Staphylococcus aureus (SA) or coagulase negative staphylococci (CNS) prosthetic joint infection (PJI) treated with debridement and retention (D/R). We calculated the treatment failure cumulative incidence (TF) of a cohort of 101 patients with SA or CNS PJI treated with D/R and antimicrobial therapy. The effect of the use of a rifampin-based regimen was evaluated. Cox proportional hazards regression evaluated the association between treatment and time-to-TF controlling for the propensity to treat with rifampin and temporal confounders. Seven percent (1/14) of the prospective rifampin-treated patients, 32% (10/31) of the historical rifampin-treated patients and 38% (21/56) of the historical non-rifampin treated patients developed TF. After controlling for the propensity to treat with rifampin and American Society of Anesthesia scores, patients in the prospective cohort had a lower risk of TF compared to patients in the historical cohort not treated with rifampin (HR 0.11; 95%CI 0.01-0.84). None (0/14) of the patients in the prospective study developed hepatotoxicity. The outcome of staphylococcal PJI treated with D/R and rifampin-based regimens was better when compared with a historical cohort treated without rifampin.

Abdominal aortic aneurysm rupture is not associated with an up-regulation of inflammation within the aneurysm wall.

BACKGROUND: The nature of the inflammatory change within ruptured AAA has not been extensively reported. The aim of this study was to compare the inflammatory response in non-ruptured and ruptured aneurysms with emphasis on the site of rupture. METHODS: Non-rupture site biopsies were taken from the anterior aneurysm sac of non-ruptured (n=31) and ruptured AAA (n=20). In 12 ruptured AAA, a further biopsy was taken from the rupture site. Enzyme-linked immunosorbent assay was used to quantify IL-6, IL-1beta and TNF-alpha. Quantitative immunohistochemistry was undertaken for generic lymphocytes, T-cells, and B-cells. RESULTS: Comparing biopsies in non-ruptured AAA versus a non-rupture site biopsy from ruptured AAA; there was no significant difference in IL-6, IL-1beta, TNF-alpha, generic lymphocytes, T-cell or B-cell content. Comparing ruptured AAA--non-rupture site with rupture site; IL-6 and TNF-alpha were unchanged. By contrast IL-1beta and lymphocytes were lower at the rupture site compared to the non-rupture site (IL-1beta 1.39 ng/mg [0.97-2.29] vs. 1.92 ng/mg [1.46-2.57], p=0.027; generic lymphocytes 2.89% [0.51-5.51] vs. 4.73% [2.27-12.40], p=0.018; T-cells 0.28% [0.04-1.18] vs. 0.82% [0.40-1.36], p=0.027; B-cells 0.16% [0.04-1.14] vs. 1.30% [0.32-5.40], p=0.021). CONCLUSIONS: These findings suggest the biological events leading to AAA rupture may not be dependent on an up-regulation in the inflammatory process.

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

Affective discrimination of stimuli that cannot be recognized.

Animal and human subjects readily develop strong preferences for objects that have become familiar through repeated exposures. Experimental evidence is presented that these preferences can develop even when the exposures are so degraded that recognition is precluded.

Evaluation of antibiotic therapy following valve replacement for native valve endocarditis.

We retrospectively evaluated 105 patients at the Mayo Clinic between 1970 and 2006 with native valve endocarditis who underwent acute valve surgery. The objective was to determine if outcomes differed based on whether they had received an antibiotic regimen recommended for native valve endocarditis or one for prosthetic valve endocarditis. Fifty-two patients had streptococcal and 53 had staphylococcal infections. Patients with each type of infection were divided into two groups: the first received postoperative monotherapy (with a beta-lactam or vancomycin), and the second received combination therapy (with an aminoglycoside for streptococcal infection, and gentamicin and/or rifampin for staphylococcal infection). The duration and types of antibiotics given pre- and postoperatively, valve cultures results, antibiotic-related adverse events, relapses, and mortality rates within 6 months of surgery were analyzed. Cure rates were similar regardless of the regimen administered. With the small number of patients in each group, a multicenter study with a larger cohort of patients is needed to better define optimal postoperative treatment regimens in this population.

Whole genome-expression profiling reveals a role for immune and inflammatory response in abdominal aortic aneurysm rupture.

OBJECTIVES: This study used the whole transcriptome approach to investigate the role of genes involved in immune and inflammatory response at the site of aneurysm rupture. MATERIALS AND METHODS: Rupture site and paired anterior sac biopsies (internal control) of ruptured abdominal aortic aneurysms (AAAs) (n=10) were analysed with Affymetrix Human Genome U133A plus 2.0 microarray. Twenty-one differentially expressed genes were selected for validation using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). RESULTS: A total of 139 genes (123 upregulated, 16 downregulated) at the aneurysm rupture site were differentially expressed (>2.5-fold, P<0.005). Immune and inflammatory responses (Gene Ontology Classification) were frequently associated with the differentially expressed genes. Genes with immune and inflammatory functions that were confirmed, by QRT-PCR, to be overexpressed at the aneurysm rupture site were interleukins-6 and -8 (IL-6 and -8), Selectin E (SELE), prostaglandin-endoperoxidase synthase 2 (COX2) and prokineticin 2 (PROK2). IL-6 (pro-immune) and IL-8 (pro-immune and pro-inflammatory) have previously been linked to aneurysm rupture; and SELE and COX2 (pro-inflammatory) have previous associations with aneurysm development but not rupture. CONCLUSIONS: The differential expression of genes involved in immune and inflammatory responses was confirmed at AAA rupture site. These genes may represent novel targets for treatment of aneurysms.

Elevated plasma MMP1 and MMP9 are associated with abdominal aortic aneurysm rupture.

BACKGROUND: The role of matrix metalloproteinases (MMPs) in abdominal aortic aneurysm (AAA) formation is well established. However the changes in plasma MMP levels with AAA rupture have not been reported. The aim of this study was to determine circulating levels of MMPs in non-ruptured and ruptured AAA immediately prior to open repair. METHODS: Concentrations of MMPs and their endogenous tissue inhibitors (TIMPs) were quantified using ELISA in pre-operative plasma samples from non-ruptured and ruptured AAA. RESULTS: MMP1 and MMP9 were elevated in the plasma of ruptured AAA versus non-ruptured AAA. A four-fold elevation in pre-operative plasma MMP9 was associated with non-survival at 30 days from rupture surgery compared with those surviving for greater than 30 days. CONCLUSION: In conclusion, these findings support the role of MMPs in AAA pathogenesis. Elevation of MMP9 was associated with ruptured aneurysm related 30-day mortality and may represent a survival indicator in this group.

G-protein-coupled inwardly rectifying potassium channels are targets of alcohol action.

G-protein-coupled inwardly rectifying potassium channels (GIRKs) are important for regulation of synaptic transmission and neuronal firing rates. Because of their key role in brain function, we asked if these potassium channels are targets of alcohol action. Ethanol enhanced function of cerebellar granule cell GIRKs coupled to GABAB receptors. Enhancement of GIRK function by ethanol was studied in detail using Xenopus oocytes expressing homomeric or heteromeric channels. Function of all GIRK channels was enhanced by intoxicating concentrations of ethanol, but other, related inwardly rectifying potassium channels were not affected. GIRK2/IRK1 chimeras and GIRK2 truncation mutants were used to identify a region of 43 amino acids in the carboxyl (C) terminus that is critical for the action of ethanol on these channels.

Regulation of blood flow in Paget's disease of bone.

Previous studies have demonstrated an increase in blood flow to extremities involved by Paget's disease of bone. It has been assumed that the increase in blood flow is through bone, but warmth of skin overlying Pagetic bone suggests that cutaneous blood flow might be increased. In three patients with Paget's disease involving one extremity, we compared blood flow in "Pagetic" extremities with flow in the contralateral normal extremities. Resting blood flow (measured with water plethysmographs) was 14.2plus or minus2.9 (meanplus or minusSE) ml/min times 100 ml extremity in the Pagetic limbs. The contribution of cutaneous flow to the increase in extremity blood flow was evaluated with epinephrine iontophoresis, which suppresses flow to skin but not to underlying tissue. Epinephrine iontophoresis of the Pagetic extremities decreased blood flow from 14.2plus or minus2.9 to 3.6plus or minus1.5 ml/min. Local heating (which increases cutaneous flow only) failed to increase blood flow in the Pagetic extremities as much as it did in the normal extremities. This suggests that cutaneous vessels in the Pagetic extremities were already dilated. During heating, blood flow in the normal extremities was similar to resting flow in the Pagetic extremities; this indicates that increases in cutaneous flow could account for most of the increase in total blood flow in the Pagetic extremities. Adrenergic control of blood flow to the Pagetic extremities was compared with that of the normal extremities. Vasoconstrictor responses to reflex stimuli in the Pagetic extremities were reduced; when vasoconstriction occurred it was gradual and sustained after termination of the stimuli, which suggests an exaggerated humoral response but reduced neural response to the stimuli. Intravenous epinephrine produced vasoconstriction in the Pagetic extremities and vasodilatation in the normal extremities. In summary, responses to epinephrine iontophoresis and heating suggest that the increase in blood flow in Pagetic extremities is primarily the result of cutaneous vasodilatation.

Selective beta-1 receptor blockade with oral practolol in man. A dose-related phenomenon.

The purpose of this study was to test the hypothesis that oral administration of a low dose of practolol in man produces selective beta-1 receptor blockade, whereas oral administration of a high dose blocks both beta-1 and beta-2 receptors. Normal men were studied 2-4 h after a single oral dose of practolol (1.5 or 12 mg/kg) and after placebo. Effects on beta-1 receptors were studied by measuring heart rate responses to exercise. Effects on beta-2 receptors were tested by measuring forearm vascular responses to brachial arterial infusions of isoproterenol. Neither dose of practolol altered base-line heart rate, forearm vascular resistance, and arterial pressure, Both low and high doses significantly attenuated heart rate responses to exercise. Forearm vasodilator responses to isoproterenol were attenuated by the high dose, but not the low dose, of practolol. Serum concentrations of practolol 2 h after administration of the drug and at the time of the studies of forearm vascular responses averaged 0.5+/-0.1 (SE) and 5.9+/-1.0 mug/ml for low and high doses of practolol, respectively. The results indicate that the phenomenon of selective beta-1 receptor blockade in man is related to the dose and serum concentration of practolol selectively block beta-1 receptors; a high dose and serum concentrations block both beta-1 and beta-2 receptors.

Effects of triiodothyronine-induced hypermetabolism on factor 8 and fibrinogen in man.

Triiodothyronine (liothyronine sodium) (400-500 mug/day for 14 days) was given to six normal subjects. Factor VIII (antihemophilic globulin) activity increased from 109 to 167% (P < 0.05); fibrinogen increased from 344 to 581 mg/100 ml (P < 0.01). To test whether the increases in factor VIII activity and fibrinogen were mediated by beta adrenergic receptors, propranolol (20 mg every 6 hr) was given orally to four other normal subjects in addition to triiodothyronine for 14 days. Factor VIII increased from 100 to 161%; fibrinogen increased from 374 to 564% (P < 0.01). Factor VIII activity did not change in a severe classical hemophiliac made hypermetabolic with triiodothyronine, but it increased from 39 to 82% in a patient with von Willebrand's disease. Triiodothyronine-induced hypermetabolism increased the incorporation of selenomethionine-(75)Se into plasma fibrinogen. These results suggest that the increases in clotting factor activity during triiodothyronine-induced hypermetabolism reflect an effect of increased protein synthesis rather than enhanced stimulation of beta adrenergic receptors.

International experts' practice in the antibiotic therapy of infective endocarditis is not following the guidelines.

OBJECTIVE: The management of infective endocarditis (IE) may differ from international guidelines, even in reference centres. This is probably because most recommendations are not based on hard evidence, so the consensus obtained for the guidelines does not represent actual practices. For this reason, we aimed to evaluate this question in the particular field of antibiotic therapy. METHODS: Thirteen international centres specialized in the management of IE were selected, according to their reputation, clinical results, original research publications and quotations. They were asked to detail their actual practice in terms of IE antibiotic treatment in various bacteriological and clinical situations. They were also asked to declare their IE-related in-hospital mortality for the year 2015. RESULTS: The global compliance with guidelines concerning antibiotic therapy was 58%, revealing the differences between theoretical 'consensus', local recommendations and actual practice. Some conflicts of interest were also probably expressed. The adherence to guidelines was 100% when the protocol was simple, and decreased with the seriousness of the situation (Staphylococus spp. 54%-62%) or in blood-culture-negative endocarditis (0%-15%) that requires adaptation to clinical and epidemiological data. CONCLUSION: Worldwide experts in IE management, although the majority of them were involved and co-signed the guidelines, do not follow international consensus guidelines on the particular point of the use of antibiotics.

Resistance of cultured Lewis lung carcinoma cell lines to tiazofurin.

An established in vitro cell line (LLTC), originally derived from the Lewis lung carcinoma (LL), was found to have lost sensitivity to the C-nucleoside antitumor agent tiazofurin (NSC-286193; 2-beta-D-ribofuranosylthiazole-4-carboxamide) both in vitro and in vivo. A new in vitro cell line (LLAK) was derived from LL and compared to LLTC in its growth properties and sensitivity to tiazofurin. LLAK resembled the in vivo tumor in having both a high S-phase fraction and a high rate of cell death at high cell density. In continuous drug exposure growth inhibition assays, the concentration of tiazofurin required to reduce the number of cells in a culture by 50% with respect to control cultures was 0.51 microM for LLAK, 2.6 microM for LLTC, and greater than 10 microM for a range of human cancer cell lines. In cytotoxicity assays involving a 2-hour drug exposure followed by clonogenic assay, tiazofurin was more toxic to LLAK cells than to LLTC cells or L1210 murine leukemia cells, consistent with its high in vivo activity against LL. MM-96 human melanoma cells were highly resistant. Flow cytometry studies indicated that tiazofurin selectively depleted the LLAK cell population of S- and G2-phase cells. In one experiment involving 16 consecutive in vitro passages of LLAK in the absence of tiazofurin, a new line emerged that was resistant to tiazofurin in the clonogenic assay. The results demonstrate the spontaneous emergence of resistance from a tiazofurin-sensitive cell line. If similar processes occur during adaptation of human tumor cells to culture, this may explain the finding of low activity of tiazofurin toward a range of human tumor cell lines.

Blood flow failure as a major determinant in the antitumor action of flavone acetic acid.

Some investigators have suggested that the marked activity of flavone acetic acid (FAA) against advanced solid tumors in mice results from an indirect effect. This study indicates that the critical effect of FAA is irreversible inhibition of tumor blood flow. Perfusion of sc Colon 38 tumors, assessed with H33342 as a fluorescent stain for functional blood vessels, was reduced to 50% of controls within 3 hours of an ip injection of 1.2 mmol of FAA/kg and was completely inhibited by 24 hours. A double-label fluorescence technique demonstrated a significant decrease in blood flow in both sc Colon 38 and im EMT-6/Ak tumors as early as 15 minutes after iv treatment with 1.2 mmol of FAA/kg, with progressively enlarging zones of perfusion failure. The rate of cell death in totally ischemic EMT-6 tumors was shown to be sufficiently rapid to represent a major component of the observed antitumor effect of FAA if the flavonoid acts via inhibition of blood flow. Further, avascular EMT-6/Ak multicellular spheroids growing in the mouse peritoneum are relatively resistant to killing by FAA administered iv or ip, despite extensive infiltration with host immune cells. These results indicate that inhibition of tumor blood flow by FAA is a necessary component of its antitumor activity against solid tumors.

Contemporary management of the infra-renal abdominal aortic aneurysm.

Abdominal aortic aneurysms (AAAs) principally affect men over 60 years of age. Aneurysms are usually asymptomatic and detected coincidentally or following the onset of symptoms. Elective repair of an AAA is considered when the diameter reaches 5.5cm or annual expansion exceeds 1 cm. Rupture represents a catastrophic event and carries an unacceptably high mortality. The advent of endovascular repair heralds an improvement in operative outcome for this disease process. In this review we provide an overview of the recent trials investigating the management of non-ruptured and ruptured aneurysms and the strategies that may be invoked to lower the mortality of this disease process

Activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide against Chinese hamster cells in multicellular spheroids.

The cytotoxic activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), a novel acridine derivative with clinical antitumor activity, has been examined in multicellular spheroids grown from Chinese hamster V79-171b cells. m-AMSA is much less effective against cells within these tumor-like structures than it is against exponential-phase V79-171b cells in monolayer cultures, the initial D0 of the survival curve for the latter being approximately 10-fold lower than for the former following a 60-min exposure to the drug. The resistance of spheroid cells to m-AMSA appears to be at least partially a result of the noncycling or slowly cycling state of the majority of these cells, although they are more sensitive than cells in plateau-phase monolayers. A further component of resistance in spheroids requires the presence of an intact spheroid structure and may be due to drug transport limitations. The use of sequential trypsinization techniques to recover cells at varying depths within spheroids demonstrates that a 60-min m-AMSA treatment preferentially kills cells nearest the spheroid surface, suggesting that tumor cells at a distance from the vasculature may limit the efficacy of m-AMSA in vivo.

Effects of amsacrine and other DNA-intercalating drugs on nuclear and nucleolar structure in cultured V79 Chinese hamster cells and PtK2 rat kangaroo cells.

Amsacrine [m-AMSA; 4'-(9-acridinylamino)methanesulfon-m-anisidide] is a synthetic intercalating agent with clinical utility in the treatment of acute leukemias and lymphomas. However, as with other intercalators, its mechanism of action is uncertain. We have examined structural changes induced by amsacrine and other intercalators (actinomycin D, Adriamycin, mitoxantrone, 9-aminoacridine) in cultured Chinese hamster (V79-171b) and rat kangaroo kidney epithelial (PtK2) cells, using light- and electron microscopy with simultaneous assessment of cell survival. During chronic exposure at low concentrations, amsacrine causes cell and nuclear enlargement, lobulation of the nucleus, and nucleolar segregation. Nucleolar segregation was also induced by the other four intercalators. The cytotoxic potency of these drugs, as measured by cell survival after 1-hr exposure, was compared with potency of induction of nucleolar segregation. Relative potencies in the two assays varied by more than 10(4)-fold, with actinomycin D the most effective and amsacrine the least effective inducer of nucleolar segregation relative to cytotoxic potency. Thus, although all five intercalators induced nucleolar segregation with high specificity, this lesion does not correlate with cell killing by these drugs. However, interference with nucleolar function (i.e., ribosomal RNA synthesis) may be responsible for the reversible cytostatic effect observed on chronic exposure to some intercalators (actinomycin D, 9-aminoacridine) at low concentrations.

Comparison of the mutagenic and clastogenic activity of amsacrine and other DNA-intercalating drugs in cultured V79 Chinese hamster cells.

The acridine derivative amsacrine (m-AMSA) is used clinically for the treatment of acute leukemias. The mutagenic activity of this drug has been evaluated at the 6-thioguanine (6-TG) and ouabain resistance loci in cultured Chinese hamster fibroblasts (V79-171b cell line). m-AMSA was found to have weak but significant mutagenic activity at the 6-TG but not at the ouabain resistance locus, after either 1- or 45-hr exposures at concentrations causing up to 90% cell kill. Two other intercalating agents with antitumor activity, Adriamycin and actinomycin D, provided essentially identical results. All three drugs were potent inducers of micronuclei in V79-171b cells, indicating high clastogenic activity. For these intercalating agents, the yield of 6-TG-resistant mutants was approximately 100-fold lower than that for ethyl methanesulfonate after exposures causing equivalent toxicity or equivalent chromosome breakage. The acridine half-mustard ICR-191 resembled ethyl methanesulfonate rather than the other intercalating agents in providing a high yield of 6-TG-resistant mutants relative to its clastogenic activity. The tumor-inactive intercalator 9-aminoacridine demonstrated only low clastogenic activity with a lack of significant mutagenic activity at toxic concentrations. These results suggest that, for m-AMSA, Adriamycin, and actinomycin D, both cell killing and mutagenesis could be direct consequences of chromosome breakage, while 9-aminoacridine may kill cells by a different mechanism. In view of its mutagenic and clastogenic activity at clinically achievable exposures and the similarity of its genotoxic properties to Adriamycin, m-AMSA should be considered a potential carcinogen.

N-[2-(2-methyl-5-nitroimidazolyl)ethyl]-4-(2-nitroimidazolyl)butanamide (NSC 639862), a bisnitroimidazole with enhanced selectivity as a bioreductive drug.

Compounds containing two redox centers, both of which must be reduced for full expression of cytotoxicity by oxygen-inhibitable pathways (bis-bioreductive drugs), have potential as cytotoxins with high selectivity for hypoxic tumor cells. The bisnitroimidazole N-[2-(2-methyl-5-nitroimidazolyl)ethyl]-4-(2-nitroimidazolyl)butanamide (NNB, NSC 639862), in which a 2-nitroimidazole and 5-nitroimidazole moiety are joined via a carboxamide linker, is highly selective for hypoxic AA8 Chinese hamster cells (200-fold by 8 h) relative to mononitroimidazoles (5-25-fold). A bis-bioreductive mechanism is consistent with the marked increase in hypoxic potency and selectivity of NNB with time and the apparent requirement that the two nitro groups be present in the same molecule. NNB differed from mononitroimidazoles in inducing fewer DNA single strand breaks at equivalent toxicity, suggesting that a duplex DNA lesion (locally doubly damaged site) may be responsible for cell killing. Alkaline elution studies and the lack of hypersensitivity of the repair-defective UV4 cell line indicate that the cytotoxic lesion is not a DNA interstrand cross-link. NNB shows greater hypoxic selectivity than the alkylating 2-nitroimidazole RB 6145 against AA8 cells and is active in combination with radiation when administered in multiple doses against the MDAH-MCa-4 mouse mammary carcinoma.