Modulatory role of rutin on 2,5-hexanedione-induced chromosomal and DNA damage in rats: validation of computational predictions.

The aim of this study was to evaluate the potentials of rutin on 2,5-hexanedione-induced toxicities. Two successive phases were involved using in silico and in vivo approaches. The in silico was adopted for potential oral toxicity and docking. The in vivo was carried-out in two stages for two weeks; the ameliorative (stage 1, first week), preventive, and curative studies (stage 2, extended to second week). In stage 1, rats were divided into four groups of seven each (distilled water, 3% (v/v) 2,5-hexanedione, 10 mg/kg rutin, and co-administration). In stage 2, the experimental groups were given either rutin or 2,5-hexanedione and treated in reverse order. Lipid peroxidation, protein carbonyl, and DNA fragmentation in tissues and bone marrow cells micronucleus were determined. The predicted Median lethal dose (LD50) of >5000 mg/kg and toxicity class of five (5) indicates the safety of rutin when orally administered. 2,5-Hexanedione comfortably docked in to the active sites of SOD (-22.857Kcal/mol; KI = 0.9621 µM), GPx (-11.2032Kcal/mol; KI = 0.9813 µM), and CAT (-16.446Kcal/mol; KI = 0.9726 µM) with strong hydrogen bond and hydrophobic interactions. However, only strong hydrophobic interaction was observed in the case of DNA (-3.3296Kcal/mol; KI = 0.9944). In vivo findings revealed deleterious effects of 2,5-hexanedione through induction of oxidative and chromosomal/DNA damage characterized by higher level of malondialdehyde, micronuclei formations, and DNA fragmentation. These have invariably, validates the findings from in silico experiments. Furthermore, rutin was able to ameliorate, protect, and reverse these effects, and was relatively non-toxic corroborating toxicity predictions. Rutin exhibited counteractive effects on 2,5-hexanedione-induced oxidative, chromosomal, and DNA damage.

Phase 2 study of imexon, a prooxidant molecule, in relapsed and refractory B-cell non-Hodgkin lymphoma.

Lymphoma cells are subject to higher levels of oxidative stress compared with their normal counterparts and may be vulnerable to manipulations of the cellular redox balance. We therefore designed a phase 2 study of imexon (Amplimexon/NSC-714597), a prooxidant molecule, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). Imexon was administered at 1000 mg/m(2) IV daily for 5 days in 21-day cycles. Gene expression analysis performed on pretreatment tumor specimens included 13 transcripts used to generate a redox signature score, previously demonstrated to correlate with lymphoma prognosis. Twenty-two patients were enrolled having follicular (n = 9), diffuse large B-cell (DLBCL) (n = 5), mantle cell (n = 3), transformed follicular (n = 2), small lymphocytic (n = 2), and Burkitt (n = 1) lymphoma. The most common grade 3/4 adverse events were anemia (14%) and neutropenia (9%). The overall response rate was 30%, including responses in follicular lymphoma (4 of 9) and DLBCL (2 of 5). Gene expression analyses revealed CD68 and the redox-related genes, GPX1 and SOD2, as well as a higher redox score to correlate with clinical responses. Therefore, pretreatment markers of oxidative stress may identify patients likely to respond to this therapeutic approach. This trial was registered at www.clinicaltrials.gov as #NCT01314014.

Chemical Reactivity and Respiratory Toxicity of the α-Diketone Flavoring Agents: 2,3-Butanedione, 2,3-Pentanedione, and 2,3-Hexanedione.

Occupational exposure to 2,3-butanedione (BD) vapors has been associated with severe respiratory disease leading to the use of potentially toxic substitutes. We compared the reactivity and respiratory toxicity of BD with that of two structurally related substitutes, 2,3-pentanedione (PD) and 2,3-hexanedione (HD). Chemical reactivity of the diketones with an arginine substrate decreased with increasing chain length (BD > PD > HD). Animals were evaluated the morning after a 2-week exposure to 0, 100, 150, or 200 ppm BD, PD, or HD (postexposure) or 2 weeks later (recovery). Bronchial fibrosis was observed in 5/5 BD and 5/5 PD rats at 200 ppm and in 4/6 BD and 6/6 PD rats at 150 ppm in the postexposure groups. Following recovery, bronchial fibrosis was observed in all surviving rats exposed to 200 ppm BD (5/5) or PD (3/3) and in 2/10 BD and 7/9 PD rats exposed to 150 ppm. Bronchial fibrosis was observed only in 2/12 HD-exposed rats in the 200 ppm postexposure group. Patchy interstitial fibrosis affected lungs of recovery groups exposed to 200 ppm PD (3/3) or BD (1/5) and to 150 ppm PD (4/9) or BD (7/10) and correlated with pulmonary function deficits. BD and PD were more reactive and produced more bronchial fibrosis than HD.

Differentiation-inducing factor-3 inhibits intestinal tumor growth in vitro and in vivo.

Differentiation-inducing factor-1 (DIF-1) produced by Dictyostelium discoideum strongly inhibits the proliferation of various types of cancer cells by suppression of the Wnt/ß-catenin signal transduction pathway. In the present study, we examined the effect of differentiation-inducing factor-3 (DIF-3), a monochlorinated metabolite of DIF-1 that is also produced by D. discoideum, on human colon cancer cell lines HCT-116 and DLD-1. DIF-3 strongly inhibited cell proliferation by arresting the cell cycle at the G0/G1 phase. DIF-3 reduced the expression levels of cyclin D1 and c-Myc by facilitating their degradation via activation of GSK-3ß in a time and dose-dependent manner. In addition, DIF-3 suppressed the expression of T-cell factor 7-like 2, a key transcription factor in the Wnt/ß-catenin signaling pathway, thereby reducing the mRNA levels of cyclin D1 and c-Myc. Subsequently, we examined the in vivo effects of DIF-3 in Mutyh(-/-) mice with oxidative stress-induced intestinal cancers. Repeated oral administration of DIF-3 markedly reduced the number and size of cancers at a level comparable to that of DIF-1. These data suggest that DIF-3 inhibits intestinal cancer cell proliferation in vitro and in vivo, probably by mechanisms similar to those identified in DIF-1 actions, and that DIF-3 may be a potential novel anti-cancer agent.

Exposure to 2,5-hexanedione is accompanied by ovarian and uterine oxidative stress and disruption of endocrine balance in rats.

2,5-Hexanedione (2,5-HD) is an aliphatic diketone identified as the main neurotoxic metabolite of the industrial chemicals n-hexane and methyl-n-butyl ketone. Considering the dearth of information on the female reproductive toxicity effects of 2,5-HD in the literature, we assessed the potential oxidative stress mechanisms of 2,5-HD in the ovary and uterus of Wistar rats. A total of 32 female rats were randomly allotted to four groups, in which rats were exposed to 2,5-HD at doses of 0% (control), 0.25%, 0.5% and 1.0% respectively in their drinking water for 21 days. The results showed that 2,5-HD significantly increased ovarian and uterine malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels (p < 0.05). Additionally, while significant decreases in ovarian catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) activities occurred in all the 2,5-HD-treated groups, uterine catalase, GST, and GPx activities increased. Further, 2,5-HD increased follicle stimulating hormone, but decreased estrogen levels in all the 2,5-HD-treated groups, while prolactin increased in the 0.5, and 1.0% 2,5-HD-treated rats compared with the control (p < 0.05). Thus, these data imply that 2,5-HD exposure disrupts hormonal homeostasis and induces oxidative stress in the ovary and uterus of rats. These findings may therefore have toxicological implications in women occupationally exposed to n-hexane and methyl-n-butyl ketone.

Inhibin B response to testicular toxicants hexachlorophene, ethane dimethane sulfonate, di-(n-butyl)-phthalate, nitrofurazone, DL-ethionine, 17-alpha ethinylestradiol, 2,5-hexanedione, or carbendazim following short-term dosing in male rats.

BACKGROUND: Inhibin B is a heterodimer glycoprotein that downregulates follicle-stimulating hormone and is produced predominantly by Sertoli cells. The potential correlation between changes in plasma Inhibin B and Sertoli cell toxicity was evaluated in male rats administered testicular toxicants in eight studies. Inhibin B fluctuations over 24 hr were also measured. METHODS: Adult rats were administered one of eight testicular toxicants for 1 to 29 days. The toxicants were DL-ethionine, dibutyl phthalate, nitrofurazone, 2,5-hexanedione, 17-alpha ethinylestradiol, ethane dimethane sulfonate, hexachlorophene, and carbendazim. In a separate study plasma was collected throughout a 24-hr period via an automatic blood sampler. RESULTS: Histomorphologic testicular findings included seminiferous tubule degeneration, round and elongate spermatid degeneration/necrosis, seminiferous tubule vacuolation, aspermatogenesis, and interstitial cell degeneration. There was a varying response of plasma Inhibin B levels to seminiferous tubule toxicity, with three studies showing high correlation, three studies with a response only at a certain time or dose, and two studies with no Inhibin B changes. In a receiver operating characteristics exclusion model analysis, where treated samples without histopathology were excluded, Inhibin B showed a sensitivity of 70% at 90% specificity in studies targeting seminiferous tubule toxicity. CONCLUSION: Decreases in Inhibin B correlated with Sertoli cell toxicity in the majority of studies evaluated, demonstrating the value of Inhibin B as a potential biomarker of testicular toxicity. There was no correlation between decreases in Inhibin B and interstitial cell degeneration. In addition, a pattern of Inhibin B secretion could not be identified over 24 hr.

Bidirectional Regulation between NDRG1 and GSK3ß Controls Tumor Growth and Is Targeted by Differentiation Inducing Factor-1 in Glioblastoma.

The development of potent and selective therapeutic approaches to glioblastoma (GBM), one of the most aggressive primary brain tumors, requires identification of molecular pathways that critically regulate the survival and proliferation of GBM. Previous studies have reported that deregulated expression of N-myc downstream regulated gene 1 (NDRG1) affects tumor growth and clinical outcomes of patients with various types of cancer including glioma. Here, we show that high level expression of NDRG1 in tumors significantly correlated with better prognosis of patients with GBM. Loss of NDRG1 in GBM cells upregulated GSK3ß levels and promoted cell proliferation, which was reversed by selective inhibitors of GSK3ß. In contrast, NDRG1 overexpression suppressed growth of GBM cells by decreasing GSK3ß levels via proteasomal degradation and by suppressing AKT and S6 cell growth signaling, as well as cell-cycle signaling pathways. Conversely, GSK3ß phosphorylated serine and threonine sites in the C-terminal domain of NDRG1 and limited the protein stability of NDRG1. Furthermore, treatment with differentiation inducing factor-1, a small molecule derived from Dictyostelium discoideum, enhanced NDRG1 expression, decreased GSK3ß expression, and exerted marked NDRG1-dependent antitumor effects in vitro and in vivo. Taken together, this study revealed a novel molecular mechanism by which NDRG1 inhibits GBM proliferation and progression. Our study thus identifies the NDRG1/GSK3ß signaling pathway as a key growth regulatory program in GBM, and suggests enhancing NDRG1 expression in GBM as a potent strategy toward the development of anti-GBM therapeutics. SIGNIFICANCE: This study identifies NDRG1 as a potent and endogenous suppressor of glioblastoma cell growth, suggesting the clinical benefits of NDRG1-targeted therapeutics against glioblastoma.

A Phase 2 Randomized, Double-Blind, Multicenter Trial of Imexon Plus Gemcitabine Versus Gemcitabine Plus Placebo in Patients With Metastatic Chemotherapy-naïve Pancreatic Adenocarcinoma.

BACKGROUND: Imexon is a cyanoaziridine-derived iminopyrrolidone which has synergistic cytotoxicity with gemcitabine. A phase 1 study of the combination demonstrated good tolerance with encouraging clinical activity and thus we conducted this randomized phase II study. MATERIALS AND METHODS: Patients with measurable, metastatic, treatment-naive pancreatic adenocarcinoma were randomized 1:1 to receive gemcitabine at 1000 mg/m days 1, 8, and 15 with either imexon, 875 mg/m or placebo days 1, 8, and 15 every 28 days. The primary endpoint was overall survival. Secondary endpoints included progression-free survival and response rate. RESULTS: A total of 142 patients were randomized, 72 to the imexon containing arm and 70 to the placebo arm. Patients in the imexon arm received an average of 3.6 cycles (range, 1 to 23) compared with 4.4 (range, 1 to 21) in the placebo arm. There was no increased rate of ≥grade 3 toxicity in the imexon arm. Seven patients had objective responses in the imexon arm (13.7%), whereas 9 did in the placebo arm (17%). In the imexon arm, 23 patients had ≥50% reduction in CA 19-9 from baseline (33%), whereas 22 did in the placebo arm (31.4%). The median progression-free survival was 2.8 months in the imexon arm (95% confidence interval [CI], 2.0-4.1 m) and 3.8 months in the placebo arm (95% CI, 2.2-4.7 m), P=0.504. The median overall survival time in the imexon arm was 5.2 months (95% CI, 4.2-6.7 m) as compared with 6.8 m (95% CI, 4.9-8.5 m) in the placebo arm, P=0.6822. CONCLUSIONS: The combination of imexon and gemcitabine does not result in improved outcome as initial therapy of metastatic pancreatic adenocarcinoma.

Imexon-based combination chemotherapy in A375 human melanoma and RPMI 8226 human myeloma cell lines.

PURPOSE: This study evaluated the cytotoxic effects of imexon (NSC-714597) in tumor cells when combined with a broad panel of chemotherapeutic drugs. METHODS: The sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays were used to analyze the degree of growth inhibition for the combination studies in the A375 human malignant melanoma and RPMI 8226 human multiple myeloma cell lines, respectively. Cells were continuously exposed to both drugs at a constant molar ratio for 4-5 days. Combination effects were analyzed using the Median Effect method. Statistical significance was inferred if the 95% confidence interval for the combination interaction (C.I.) values for a particular two-drug combination did not include 1.0 (additivity). Synergy was inferred for C.I. values<1.0 and antagonism for CI values>1.0. RESULTS: Imexon was synergistic when combined with DNA-binding agents (cisplatin, dacarbazine, melphalan) and pyrimidine-based antimetabolites (cytarabine, fluorouracil, gemcitabine) in both cell lines. Antagonistic combinations with imexon included methotrexate and the topoisomerase I (TOPO I) and II (TOPO II) inhibitors irinotecan, doxorubicin, mitoxantrone and etoposide. Docetaxel was synergistic with imexon in both cell lines whereas paclitaxel and fludarabine showed a mixed result. Dexamethasone and the proteasome inhibitor bortezomib showed synergy in myeloma cells and additivity in the melanoma cells. The vinca alkaloid, vinorelbine, and the multi-targeted antifol, pemetrexed, were additive with imexon in both cell lines. DISCUSSION: The consistent synergy seen for imexon and alkylating agents may relate to the sulfhydryl-lowering effect of imexon, which would render cells more sensitive to electrophilic species from the alkylators. The marked synergy noted with pyrimidine-based antimetabolites was unexpected and may relate to the induction of cell cycle arrest in S-phase. The strong antagonism noted for imexon with topoisomerase I and II inhibitors may be due to the effect of imexon at increasing oxidant levels which are known to antagonize the cytotoxic effects of topoisomerase poisons. In contrast, the synergy seen with bortezomib in myeloma cells may be related to an increase in reactive oxygen species (ROS) from both drugs. These results suggest that combinations of imexon with alkylating agents and pyrimidine-based antimetabolites are rational to pursue in therapeutic studies in vivo.

New analytical techniques to facilitate preformulation screening in propellant systems.

The objective of these studies was to investigate the applicability of an online direct inject HPLC method for the preformulation screening of pharmaceutical agents in pressurized metered dose inhalers (MDIs). The technique was initially utilized for the solubility determination of solid solutes. This study explores the extension of the online direct inject method for the evaluation of drug stability in propellant systems as well as for the analysis of MDI vials crimped with metered valves. Through-life content analysis confirmed that a single vial may be repeatedly sampled, thus facilitating the stability evaluation of a single unit over time. The method was successfully used for evaluating the stability of a model drug, as a function of several different formulation configurations, with minimal sample numbers. Additionally, studies determined that after modifications were made to the injection coupler, the technique was also feasible for use with 50 and 100 microL metered valves, however further modifications are necessary for 25 microL valves.

Oxidative stress and apoptosis: a new treatment paradigm in cancer.

Redox regulation has been shown to be an important component of malignant cell survival. Tipping the cellular redox balance through pharmacologic regulation in favor of increasing intracellular reactive oxygen species (ROS) and/or depleting protective reducing metabolites (such as glutathione and nicotinamide adenine dinucleotide phosphate) may lead to oxidative stress and resultant induction of apoptosis for the treatment of cancer. We review the biology and importance of ROS with regard to malignant and normal cells. Moreover, we discuss pre-clinical and clinical data regarding novel therapeutic agents that modulate the cellular redox system including buthionine sulfoximine, ascorbic acid, arsenic trioxide, imexon, and motexafin gadolinium as single-agents and in combination. Continued research is needed to better understand the mechanisms and specific apoptotic pathways involved in ROS-induced cell death, as well as, to determine the most rationale and effective combination of redox-active agents.

2,5-Hexanedione-induced changes in the neurofilament subunit pools of rat peripheral nerve.

Axon atrophy is the principle morphological feature of the peripheral neuropathy induced by 2,5-hexanedione (HD). Axon caliber is determined by a stationary neurofilamentous cytoskeleton that is maintained through dynamic interactions with mobile neurofilament (NF) subunits. To determine the effects of HD on the stationary and mobile NF pools, groups of rats were exposed to HD at dosing schedules (175 mg/kg x 101 days or 400 mg/kg x 26 days) that produced moderate levels of neurological deficits and, as assessed by previous studies, prevalent axon atrophy in peripheral nerve. Sciatic and tibial nerves from HD-intoxicated rats and their age-matched controls were triton-extracted and separated by differential centrifugation into a high-speed pellet (P1) of NF polymer and a corresponding supernatant fraction (S1), which presumably contained mobile monomer. Cytoskeletal proteins (NF-L, NF-M, NF-H and beta-tubulin) in each fraction were determined by immunoblot analysis. Results show that regardless of HD dose-rate, triton-soluble NF subunits in the supernatant fractions were significantly reduced, whereas triton-insoluble proteins in the corresponding pellets were inconsistently affected. Beta-tubulin also exhibited inconsistent fractional changes, while abnormal higher molecular weight NF proteins were detected primarily in the triton-insoluble fraction. Studies with antibodies directed against phosphorylated (RT97) and non-phosphorylated (SMI32) epitopes on NF-H did not reveal major changes in subunit phosphorylation. These results suggest that HD intoxication is primarily associated with depletion of soluble NF proteins, which could produce axon atrophy through disruption of cytoskeletal turnover and maintenance.

Pharmaceutical development of a lyophilised dosage form for the investigational anticancer agent Imexon using dimethyl sulfoxide as solubilising and stabilising agent.

Imexon is a member of the class of 2-cyanoaziridine derivatives, which have been of interest as immunomodulators and anticancer agents since the late 1970s. For the scheduled phase I clinical trials a stable, sterile, injectable pharmaceutical dosage form containing 100 mg Imexon was required. Despite adequate solubility, its instability in aqueous media seriously hampered the pharmaceutical development of Imexon. In this study we describe the successful use of the organic solvent dimethyl sulfoxide (DMSO) as a formulation vehicle for Imexon. DMSO is shown to provide the stability required for Imexon during manufacturing and to be a suitable vehicle for lyophilisation, which was employed to gain sufficient shelf-life for the final product. The relatively low vapour pressure of DMSO, which would theoretically result in extremely slow sublimation during lyophilisation, was shown not to limit the successful lyophilisation of Imexon from DMSO at a concentration of 25 mg/mL. The lyophilisation cycle developed for Imexon resulted in residual DMSO contents of 4.6 +/- 0.6% in the lyophilised product, limiting the amount of DMSO administered to the patient to well below the 50 mg/day acceptable in pharmaceutical products as stated in ICH guidelines. Imexon 100 mg/vial lyophilised product was shown stable for at least 12 months of storage at -20 degrees C and +5 +/- 3 degrees C in the dark.

Effects of male reproductive toxicants on gene expression in rat testes.

Predictive biomarkers of testicular toxicity are needed for an efficient development of drugs. The purpose of the present study was to obtain further insight into the toxicity mechanisms of various male reproductive toxicants and to detect genomic biomarkers for rapid screening of testicular toxicity. Four reproductive toxicants, 2,5-hexanedione (Sertoli cells toxicant), ethylene glycol monomethyl ether (EGME; spermatocytes toxicant), cyclophosphamide (spermatogonia toxicant) and sulfasalazine, were orally administered to male rats once. Six hours after the single dosing, gene expression in the testes was monitored by cDNA microarray and real-time RT-PCR and the testes were histopathologically examined. No histopathological abnormality was detected except for slight degeneration of spermatocytes in the EGME-treated testes. cDNA microarray analysis revealed differential gene expression profiles, and it was possible based on the profiles to characterize the action of the compounds in the testes. Interestingly, 3 spermatogenesis-related genes -- heat shock protein 70-2, insulin growth factor binding protein 3 and glutathione S transferase pi -- were affected by all the compounds. The above changes of gene expression were detectable within a short period after the dosing prior to the appearance of obvious pathological changes. These data suggest that cDNA microarray is a useful technique for evaluation of primary testicular toxicity. Furthermore, we propose the above 3 spermatogenesis-related genes as potential biomarkers of testicular toxicity.

2,5-hexanedione and carbendazim coexposure synergistically disrupts rat spermatogenesis despite opposing molecular effects on microtubules.

2,5-Hexanedione (2,5-HD), a taxol-like promoter of microtubule assembly, and carbendazim (CBZ), a colchicine-like inhibitor of microtubule assembly, are two environmental testicular toxicants that target and disrupt microtubule function in Sertoli cells. At the molecular level, these two toxicants have opposite effects on microtubule assembly, yet they share the common physiologic effect of inhibiting microtubule-dependent functions of Sertoli cells. By studying a combined exposure to 2,5-HD and CBZ, we sought to determine whether CBZ would antagonize or exacerbate the effects of an initial 2,5-HD exposure. In vitro, 2,5-HD-treated tubulin had a decreased lag time and an increased maximal velocity of microtubule assembly. These 2,5-HD-induced in vitro alterations in microtubule assembly were normalized by CBZ exposure. In vivo, adult male rats were exposed to a 1% solution of 2,5-HD in the drinking water for 2.5 weeks. CBZ was administered by gavage (200 mg/kg body weight) at the same time as unilateral surgical ligation of the efferent ducts, 24 h before evaluation of the testis. Measures of testicular effect (testis weight, histopathologic changes [sloughing and vacuolization], and seminiferous tubule diameters) were all significantly altered with combined exposure. The testicular effects in the combined exposure group were either different (seminiferous tubule diameters), additive (% vacuolization), or greater than additive (% sloughing) compared to the effects of the individual toxicant exposure groups referenced to the controls. Therefore, CBZ coexposure does not antagonize the effects of an initial 2,5-HD exposure, as might be expected if their molecular effects on microtubule assembly were solely responsible for their combined toxicity; instead, 2,5-HD and CBZ act together to exacerbate the testicular injury.

Redefining toxic distal axonopathies.

Nerve damage classified as a central-peripheral distal axonopathy is produced by a variety of chemicals (e.g. acrylamide, n-hexane). Historically, axon swelling and secondary degeneration have been considered the morphologic hallmarks of toxic axonopathies and substantial research has been devoted toward deciphering corresponding molecular mechanisms. However, recent studies from the author's laboratory investigating rate (mg toxicant/kg/day) and route (i.p. vs gavage) of intoxication have shown that swelling and degeneration were related to neurotoxicant dosing conditions (i.e. low-dose, subchronic exposure) and not to development of neurophysiological deficits or classic behavioral toxicity. This suggests the presumed hallmarks of distal axonopathy are epiphenomena of uncertain pathophysiologic significance. Therefore, the current definition of and chemical classification scheme for toxic distal axonopathies requires re-evaluation.

An electrophysiological study of 2-hexanone and 2,5-hexanedione neurotoxicity in rats.

n-Hexane and its metabolites are neurotoxic to animals and man. Studies have revealed a progressive neuropathy which affects the distal regions of motor and sensory peripheral nerves. This paper describes efforts to determine whether 2-hexanone or 2,5-hexanedione is more neurotoxic than 2-hexanone and that it first affects the distal axon. Concentrations of 20 mM produced no effects after 3 weeks but 40 mM increased distal latency after 2 weeks.

Thermolabile hemoglobin formation by 2-cyanaziridine derivatives.

The erythrocyte toxicity of imexone, azimexone, ciamexone, and its derivatives was assessed by means of the thermolabile hemoglobin formation test. After administration of doses up to 500 mg/kg to mice, imexone had no effect, whereas in accordance with previous studies azimexone strongly and ciamexone moderately enhanced thermolabile hemoglobin formation. With regard to the derivatives (metabolites) of ciamexone, the alcohol was as active as ciamexone itself, whereas ciamexone aldehyde gave a weak reaction. Both ciamexone acid and ciamexone-cysteine had no influence. Demethyl ciamexone, however, was more toxic than ciamexone, further indicating that the metabolic oxidation of the methyl group represents a detoxifying step. In contrast to the in vivo results, after incubation with blood samples solely imexone (in the range of mg/ml blood) but none of the other 2-cyanaziridines enhanced thermolabile globin formation. Preincubation of imexone with cysteine inhibited this effect. The results are discussed in view of the hypothesis that binding of the nitrile group of the 2-cyanaziridines to mercapto groups of proteins precedes their biological effects.

Total number and mean cell volume of neocortical neurons in rats exposed to 2,5-hexanedione with and without acetone.

The toxicological effects of 2,5-hexanedione (2,5-HD) alone and combined with acetone on the number and size of neurons in the cerebral cortex of rats were evaluated with stereological techniques. Thirty rats were equally divided into three groups: One control, one receiving 0.5% 2,5-HD, and one receiving 0.5% 2,5-HD and 0.5% acetone in the drinking water for seven weeks. Unbiased estimates of the total number of neocortical neurons, as well as the mean neuronal nuclei and cell body volumes were obtained from systematically sampled 3.5-microns sections. The total number of neurons in the 2,5-HD group was significantly smaller than the control group (p less than 0.05, one-tailed t-test). Both test groups showed significant changes in the mean cell body volume: Compared with the control group, animals exposed to 2,5-HD had 11% smaller cell body volumes while animals exposed to 2,5-HD and acetone had 13% larger cell body volumes. These data represent the first unbiased estimation of mean cell volume in toxicology. We propose the nucleator method as an efficient and accurate tool for estimating quantitative changes in toxicological research.

Effects of 2,5-hexanedione alone and in combination with acetone on radial arm maze behavior, the "brain-swelling" reaction and synaptosomal functions.

A multidisciplinary study to investigate 2,5-hexanedione (2,5-HD)-induced CNS-neurotoxicity and its proposed potentiation by acetone (AC) was conducted. Neurobehavioral testing, estimation of the brain-swelling reaction, neuropathological and morphometric measurements as well as synaptosomal 5-hydroxytryptamine (5-HT) uptake were evaluated. Male Wistar rats were exposed through their drinking water, for seven weeks, either to 0.5% 2,5-HD alone or to 0.5% 2,5-HD in combination with 0.5% AC. This 2,5-HD dose is known to cause neurotoxicity in the peripheral nervous system. Exposed animals were compared to a control group that received tap water. Acquisition but not performance of spatial learning, as measured in the radial 8-arm maze, was significantly inhibited by the coexposure to 2,5-HD and AC. Brain weights of rats exposed to 2,5-HD alone and to 2,5-HD and AC in combination, were slightly, albeit significantly, reduced. Synaptosomal high-affinity 5-HT uptake rate and uptake capacity were significantly reduced by 2,5-HD alone and in combination with AC. The morphometric results are reported in a companion paper. In conclusion, the present findings indicate that 2,5-HD is a CNS-neurotoxicant. The hypothesis about AC potentiation of 2,5-HD CNS-neurotoxicity was supported.