A silk peptide fraction restores cognitive function in AF64A-induced Alzheimer disease model rats by increasing expression of choline acetyltransferase gene.

This study investigated the effects of a silk peptide fraction obtained by incubating silk proteins with Protease N and Neutrase (SP-NN) on cognitive dysfunction of Alzheimer disease model rats. In order to elucidate underlying mechanisms, the effect of SP-NN on the expression of choline acetyltransferase (ChAT) mRNA was assessed in F3.ChAT neural stem cells and Neuro2a neuroblastoma cells; active amino acid sequence was identified using HPLC-MS. The expression of ChAT mRNA in F3.ChAT cells increased by 3.79-fold of the control level by treatment with SP-NN fraction. The active peptide in SP-NN was identified as tyrosine-glycine with 238.1 of molecular weight. Male rats were orally administered with SP-NN (50 or 300mg/kg) and challenged with a cholinotoxin AF64A. As a result of brain injury and decreased brain acetylcholine level, AF64A induced astrocytic activation, resulting in impairment of learning and memory function. Treatment with SP-NN exerted recovering activities on acetylcholine depletion and brain injury, as well as cognitive deficit induced by AF64A. The results indicate that, in addition to a neuroprotective activity, the SP-NN preparation restores cognitive function of Alzheimer disease model rats by increasing the release of acetylcholine.

Involvement of a volatile metabolite during phosphoramide mustard-induced ovotoxicity.

The finite ovarian follicle reserve can be negatively impacted by exposure to chemicals including the anti-neoplastic agent, cyclophosphamide (CPA). CPA requires bioactivation to phosphoramide mustard (PM) to elicit its therapeutic effects however; in addition to being the tumor-targeting metabolite, PM is also ovotoxic. In addition, PM can break down to a cytotoxic, volatile metabolite, chloroethylaziridine (CEZ). The aim of this study was initially to characterize PM-induced ovotoxicity in growing follicles. Using PND4 Fisher 344 rats, ovaries were cultured for 4 days before being exposed once to PM (10 or 30 µM). Following eight additional days in culture, relative to control (1% DMSO), PM had no impact on primordial, small primary or large primary follicle number, but both PM concentrations induced secondary follicle depletion (P<0.05). Interestingly, a reduction in follicle number in the control-treated ovaries was observed. Thus, the involvement of a volatile, cytotoxic PM metabolite (VC) in PM-induced ovotoxicity was explored in cultured rat ovaries, with control ovaries physically separated from PM-treated ovaries during culture. Direct PM (60 µM) exposure destroyed all stage follicles after 4 days (P<0.05). VC from nearby wells depleted primordial follicles after 4 days (P<0.05), temporarily reduced secondary follicle number after 2 days, and did not impact other stage follicles at any other time point. VC was determined to spontaneously liberate from PM, which could contribute to degradation of PM during storage. Taken together, this study demonstrates that PM and VC are ovotoxicants, with different follicular targets, and that the VC may be a major player during PM-induced ovotoxicity observed in cancer survivors.

Changes in activity of proline-specific peptidases in rat model for dementia of Alzheimer's type.

We studied the role of proline-specific peptidases in the pathogenesis of Alzheimer's disease. Testing of conditioned passive avoidance 24 h after learning showed that chronic administration of scopolamine to rats 4-fold reduced the latency of entry into the dark chamber in comparison with controls (intact animals). Activity of prolyl endopeptidase was significantly higher than in the controls in both the cortex and hippocampus. Changes in dipeptidyl peptidase IV activity were observed only in the cortex. Injection of AF-64A toxin into Meynert nucleus basalis reduced the latency of entry into the dark compartment by 75% in comparison with that in sham-operated and intact controls. Prolyl endopeptidase activity was reduced in the frontal cortex and hippocampus, but not in hypothalamus. Changes in dipeptidyl peptidase IV activity were detected only in the frontal cortex.

In vitro cytotoxicity study of oxaziridines generated after chlordiazepoxide, demoxepam, and desmethylchlordiazepoxide UV irradiation.

The cytotoxicity of oxaziridines photogenerated after irradiation of chlordiazepoxide (CDZ) and its metabolites was investigated in vitro by a MTT assay on P388 leukemia and B16 melanoma cell lines and compared with that of the anticancer drug, melphalan. For the longer time-exposure experiment, oxaziridines had the same cytotoxicity as melphalan and this toxicity was higher when oxaziridines were photogenerated in the presence of cells. In conclusion, oxaziridines generated after CDZ, demoxepam, and desmethylchlordiazepoxide ultraviolet irradiation exhibited cytotoxicity activity against cancer cell lines. A possibility of CDZ use within the context of photodynamic therapy as a treatment for small, superficial tumors should not be excluded, because oxaziridines can be generated locally by skin-tumor local irradiation after CDZ topical administration.

First synthesis of N-[(aziridin-2-yl)methyl]benzimidazolequinone and analysis of toxicity towards normal and Fanconi anemia cells.

A diazole is N-substituted with 1-trityl-2-methylaziridine and demethylated and oxidised with NBS under acidic conditions to give a benzimidazolequinone; this novel anti-tumour agent is marginally more cytotoxic than mitomycin C (MMC) towards the normal human fibroblast cell line GM00637, while the MMC-hypersensitive human Fanconi anaemia (FA) cell line, PD20i, lacking the FANCD2 protein, is also hypersensitive to the benzimidazolequinone, with expression of FANCD2 protein decreasing sensitivity to both MMC and the benzimidazolequinone.

[Estimation of the frequencies of induced mutations in spermatogenic cells of senescence-accelerated prone mice of the SAMP1 strain].

The results obtained in this work demonstrate the dynamics of cytogenetic changes of spermatogenic cells in senescence-accelerated prone mice, strain SAMP1, after a single exposure to a chemical mutagen, dipin, at a genetically active dose of 30 mg/kg. In the time interval between days 3 and 28 the frequency of induced spermatogonial micronuclei does not significantly exceed the level of spontaneous mutagenesis. The lack of an experimental effect of micronuclei in this time interval is probably a consequence of mitotic delay and (or) of the death of a considerable part of genetically defective cells in the spermatogonial compartment. Different stages of meiosis exhibit different chemical sensibilities: the yield of round spermatids with micronuclei is maximum after treatment of early primary spermatocytes (preleptotene-leptotene stage) with dipin. The high sensibility of preleptotene and leptotene spermatocytes is confirmed by the sperm head shape abnormality assay. Chromosome damage caused by dipin in spermatogonial stem cells is irreversible, as evidenced by a sharp increase in the frequencies of spermatogonial and meiotic micronuclear aberrations within long periods after treatment. Increased genetic instability in the stem compartment does not lead to irreversible degradation of the system of development of male sex cells in senescence-accelerated SAMP1 mice.

[Response of the spermatogenic system to chemical mutagen dipin in SAMP1 senesce-accelerated mice].

Specific features of spermatogenesis were studied in senesce-accelerated mice of the line SAMP-1 after one-time injection of the chemical mutagen dipin. Quantitative and histomorphological changes in the spermatogenic epithelium proved to develop gradually. Cell loss and disorganization of spermatogenesis reached the peak as late as on days 28 and 35 after the injection. Differentiating spermatogonia manifested increased sensitivity to dipin. In prophase I of meiosis, developing spermatocytes proved to be less sensitive to the cytotoxic action of dipin at the pachytene than at the preleptotene-leptotene stages. Spermatogenesis in most seminiferous tubules was restored by day 56 after dipin treatment. At the end of the experiment (day 100), both quantitative parameters and morphological pattern of spermatogenesis did not differ significantly from those in the control. Thus, the cytotoxic action of dipin does not lead to irreversible structural disorganization of the spermatogenic epithelium in SAMP1 mice. Radioautography revealed a large proportion of highly differentiated Sertoli cells with 3H-thymidine-labeled nuclei in experimental animals. In some cases, structures resembling embryonic seminiferous tubules were revealed in the vicinity of rete testis in testis sections of experimental mice. These structures contained the cells morphologically similar to gonocytes and young Sertoli cells.

Hepatic nitroreduction, toxicity and toxicokinetics of the anti-tumour prodrug CB 1954 in mouse and rat.

5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954), a promising anti-tumour compound, is associated with clinical hepatotoxicity. We have previously demonstrated that human liver preparations are capable of endogenous 2- and 4-nitroreduction of CB 1954 to generate highly potent cytotoxins. The present study initially examined the in vitro metabolism of CB 1954 in S9 preparations of several non-clinical species and strains. The CD-1 nu/nu mouse and Sprague-Dawley rat were subsequently chosen for further assessment of in vivo metabolism and hepatotoxicity of CB 1954, as well as the mechanisms that may be involved. Animals were administered the maximum tolerated dose (MTD). At 562 micromol/kg, the mouse exhibited transaminase elevation and centrilobular hepatocyte injury. Moreover, thiol adducts as well as hepatic glutathione depletion paralleled temporally by maximal nitroreduction were observed. The rat had a much lower MTD of 40 micromol/kg and showed signs of gastro-intestinal disturbances. In contrast to mouse, peri-portal damage and biliary changes were observed in rat without any alterations in plasma biomarkers or hepatic glutathione levels. Immunohistochemical analysis did not reveal any correlation between the location of injury and expression of cytochrome P450 reductase and NAD(P)H quinone oxidoreductase 1, two enzymes implicated in the bioactivation of this drug. In conclusion, the present study showed that following administration of CB 1954 at the respective MTDs, hepatotoxicity was observed in both mouse and rat. However, the degree of sensitivity to the drug and the mechanisms of toxicity involved appear to be widely different between CD-1 nu/nu mice and Sprague-Dawley rats.

Carcinogenicity tests of certain environmental and industrial chemicals.

Fourteen chemicals of varied uses were tested for carcinogenicity by oral administration in male and female Charles River CD rats. Under the conditions of the tests, propane sultone, propylene imine, and ethylenethiourea, in addition to the positive control N-2-fluorenylacetamide, were carcinogenic. Avadex, bis(2-chloroethyl) ether, the potassium salt of bis(2-hydroxyethyl) dithiocarbamic acid, ethylene carbonate, and semicarbazide hydrochloride were not carcinogenic under the test conditions. Dithiooxamide, glycerol alpha-monochlorohydrin, and thiosemicarbazide gave somewhat ambiguous results, though administered at high enough dose levels to be toxic. An inadequate number of animals survived treatments with sodium azide, sodium bisulfide, and vinylene carbonate, or the animals may not have received sufficiently high doses of the test chemicals to provide maximum test sensitivity. However, there were no indications that these three chemicals were carcinogenic under the test conditions.

Induction of DNA strand breaks and cross-links by 2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone in Chinese hamster ovary cells.

The DNA-damaging effects of 2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone (AZQ) in Chinese hamster ovary cells were investigated. As determined by alkaline elution, DNA strand breaks were observed in cells treated with 50 microM AZQ for 2 hr. The single-strand break frequency was 31.3 +/- 5.3 (S.D.) rad equivalents. Strand breaks could also be detected at lower drug concentration if proteinase K treatment was included before DNA elution. In comparison, DNA cross-links were apparent in cells treated with as low as 6.25 microM AZQ. The cross-linking frequencies were 39.7, 124.3, 230.3, and 625.1 rad-equivalents for 6.25, 12.5, 25, and 50 microM AZQ, respectively. Both DNA-DNA and DNA-protein cross-links in AZQ-treated cells were revealed by the proteinase K assay. The DNA strand breaks induced by AZQ were rapidly rejoined within 1 hr after drug removal. DNA interstrand cross-links increased within the first 6 hr of postincubation and then slightly decreased by 12 hr, and most of the cross-links disappeared after cells were allowed to recover for 24 hr. DNA-protein cross-links were immediately formed during the drug treatment period and were gradually decreased after drug removal.

Comparison of the structural and cytotoxic activity of novel 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone analogues.

Eight analogues of 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone have been synthesized and tested for cytotoxicity against four different leukemic and lymphomic cell lines. For K562 and BSM cells, the toxicity could be correlated with the ease of reduction of the compounds as determined by the one-electron reduction potentials and the electron spin resonance detection of the reduced compounds produced by the cells. The cell toxicity could also be correlated with the efficiency of the compounds to form cross-links in DNA. However, no such correlations could be observed for the L1210 and Raji cells, although the activity of the NADPH dependent reducing enzymes in these cells was similar to that in the others. It is believed that for the L1210 and Raji cells, the influx/efflux of the different compounds may be more important to the cytotoxicity than their reduction or alkylation.

Decreased cytotoxicity of aziridinylbenzoquinone caused by polyamine depletion in 9L rat brain tumor cells in vitro.

The in vitro cytotoxicity of aziridinylbenzoquinone (AZQ) used either alone or after induced intracellular polyamine depletion in 9L rat brain tumor cells was studied using a colony-forming efficiency assay. Used alone, AZQ was cytotoxic to 9L cells; however, depletion of intracellular putrescine and spermidine levels by treatment with 1 mM alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, for 72 hr decreased significantly the cytotoxicity of AZQ. Dose modification factors were 1.9 and 1.8 at 10 and 1% survival levels, respectively. Decreased cytotoxicity could be almost completely prevented by addition of putrescine to polyamine-depleted cells 24 hr before AZQ treatment. Although AZQ alone was cytotoxic against 9L cells, metabolic activation by the S-9 rat liver microsomal fraction increased greatly the observed cytotoxicity. However, even with microsomal activation, pretreatment of cells with 1 mM alpha-difluoromethylornithine for 48 hr produced a significant decrease in AZQ cytotoxicity; dose modification factors were 2.4 and 2.2 at 10 and 1% survival levels, respectively. Addition of putrescine to polyamine-depleted cells 24 hr before AZQ treatment prevented the decrease in cytotoxicity. Pretreatment of 9L cells for 48 hr with 40 microM methylglyoxal bis(guanylhydrazone), a polyamine biosynthesis inhibitor that competitively inhibits S-adenosylmethionine decarboxylase, caused a decrease in the cytotoxicity of AZQ administered without microsomal activation. The dose modification factor was 1.6 at both 10 and 1% survival levels.

Loss in cell killing effectiveness of anticancer drugs in human gastric cancer clones due to recovery from potentially lethal damage in vitro.

The ability of human gastric cancer clones to recover from potentially lethal damage was studied. Recovery was greatest following treatments with bleomycin or Adriamycin; the recovery ratios (i.e., survival) increased almost 8-fold during a posttreatment incubation period. Recovery was also possible following treatments with actinomycin D, 1,2:5,6-dianhydrogalactitol, and diaziquone; however, the recovery ratios never increased above 2. No recovery was observed following treatment with 5-fluorouracil. Recovery from potentially lethal damage may be related to the heterogeneity in survival responses observed following treatment with some anticancer drugs. Bleomycin and Adriamycin treatments result in large heterogeneous survival fractions among these human stomach cancer clones, and the potentially lethal damage recovery ratios were larger (and variable). However, actinomycin D, diaziquone, and 1,2:5,6-dianhydrogalacticol produce very uniform killing effects in these cells and the recovery ratios are very much smaller and less variable. Finally the large amount of recovery observed after bleomycin or Adriamycin treatments resulted in the loss of cell killing effectiveness of the agents. Because the survival fractions increased during the recovery period, the net effect on cell killing was reduced to an amount normally obtained with doses that were up to six times smaller.

Cellular pharmacology of N1- and N8-aziridinyl analogues of spermidine.

We have previously described the synthesis and cytotoxic properties of 2 polyamine analogues in which either the N1- or N8-amino group of spermidine was replaced by the alkylating moiety, aziridine. However, the mechanisms by which these aziridinyl analogues of spermidine inhibit cell growth remain unknown. As a result, we have studied: (a) the effect of pretreatment with difluoromethyl ornithine (DFMO) and coincubation with exogenous spermidine on cytotoxicity induced by the aziridinyl spermidines; (b) the reversibility of the cytotoxicity induced by the aziridinyl spermidines; (c) the accumulation of N1- and N8-aziridinyl spermidine by cells and the effects of DFMO on this process; and (d) the impact of N1- and N8-aziridinyl spermidine on cellular polyamine pools and on cellular accumulation of spermidine. The cytotoxicity induced by these 2 aziridinyl derivatives of spermidine [concentration required to inhibit cell growth or incorporation of radiolabeled precursor into trichloroacetic acid-precipitable material by 50% (IC50) N1 = 0.2 microM, IC50 N8 = 0.4 microM)] was potentiated by pretreatment of L1210 cells for 24 h with 100 microM DFMO (IC50 N1 = 0.05 microM, IC50 N8 = 0.15 microM) and was prevented by coincubation with 3.7 microM spermidine (IC50 N1 = 1.1 microM, IC50 N8 = 2.4 microM). In contrast, similar pretreatment with DFMO or coincubation with spermidine had no effect on the cytotoxicity induced by the aziridine-containing alkylating agent, N,N',N"-triethylenethiophosphoramide (thiotepa) (IC50 = 2.4 microM). The cytotoxicity induced by 24-h incubation with either N1- or N8-aziridinyl spermidine was not altered by removal of those compounds and incubating treated cells in medium augmented with 3.7 microM spermidine. However, and as expected, similar maneuvers did not reverse the cell growth-inhibitory effect induced by 24-h incubation with 100 microM DFMO. Cellular accumulation of both N1- and N8-aziridinyl spermidine increased with increasing extracellular concentrations. N1-Aziridinyl spermidine was accumulated to a greater degree than was the N8-analogue, achieving up to 6-fold higher intracellular concentrations at the same extracellular concentration. Cellular accumulation of both aziridinyl compounds was greatly enhanced by 24-h pretreatment with DFMO. Both N1- and N8-aziridinyl spermidine inhibited the uptake of spermidine in a dose-dependent manner. The perturbation of polyamine biochemistry by the test compounds was characterized by their ability to deplete cellular putrescine, as well as spermidine and spermine. These results imply that the cytotoxic mechanism of the aziridinyl spermidine analogues is, to a great extent, dependent on their polyamine nature and may imply selectivity for rapidly growing and neoplastic cells.

Topical chemotherapy of intradermal Walker 256 carcinosarcoma with diaziquone and doxorubicin in the rat.

A model for metastatic skin cancer using intradermal injection of Walker 256 carcinosarcoma has been developed in the rat. Using this model, antitumor activity of topically applied doxorubicin and diaziquone in Vanicream, Plastibase, and dimethyl sulfoxide (DMSO) as vehicles was compared with intraperitoneal injection of the drugs at the same doses beginning 4 days after injection of tumor cells. Doxorubicin applied topically at 0.5 mg/day for 4 days in Vanicream or Plastibase exhibited no antitumor activity, while i.p. administered doxorubicin at 0.5 mg/day for 4 days inhibited tumor growth at day 20 by 66%. Diaziquone applied topically at 0.1 mg/day for 4 days in Vanicream, Plastibase, or DMSO inhibited tumor growth at day 20 by 66, 86, and 43%, respectively, and cured animals of the skin tumor at a dose of 0.5 mg/day. Diaziquone administered i.p. at 0.5 mg/day for 4 days was lethal to rats, and at 0.1 mg/day it produced 93% inhibition of tumor growth at day 20. Diaziquone applied topically at 0.1 mg/day for 4 days in Plastibase cured rats of advanced tumor when treatment was begun 12 days after injection of tumor cells. The area under the plasma radioactivity time curve over 5 h for a single 0.64-mg dose of topically applied [ring-14C]diaziquone in DMSO was 0.01% that of the same dose of [ring-14C]diaziquone administered i.p. in non-tumored rats. The decrease in WBC count following topical application of diaziquone at a dose of 0.1 mg/day for 4 days, compared to the same dose of diaziquone administered i.p., was 62% in Vanicream, 81% in Plastibase and 33% in DMSO. Topical diaziquone was non-toxic to normal skin in the rat and in the domestic pig. It is concluded that topical application of diaziquone offers a therapeutic advantage over systemic treatment for metastatic cancer of the skin.

DNA strand scission and cross-linking by diaziridinylbenzoquinone (diaziquone) in human cells and relation to cell killing.

The effects of 3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone (diaziquone; AZQ) on various cell types were studied in relation to two chemical reactivities that this drug would be expected to have intracellularly. AZQ can undergo a reduction-oxidation cycle of the quinone function; this could generate free radicals which could produce DNA damage, especially DNA strand scission. The second reactivity, based on the two aziridine groups, could produce alkylation reactions that could produce DNA cross-links. DNA strand breakage and cross-linking were measured by alkaline elution and were compared with cell killing assayed by colony survival. Among the cell strains studied (human IMR-90, VA-13, and HT-29 and mouse L1210), marked differences were found in the magnitudes of DNA strand breakage and interstrand cross-linking produced by AZQ. Most striking, IMR-90 cells exhibited substantial strand breakage and little or no interstrand cross-linking, whereas the reverse was true for HT-29 cells. Cell killing correlated well with interstrand cross-linking but did not correlate with strand scission in these cell lines. It is concluded that AZQ produces DNA strand breaks and interstrand cross-links by different mechanisms which vary independently among different cell lines.

Treatment of human glioma and medulloblastoma tumor lines in athymic mice with diaziquone and diaziquone-based drug combinations.

We used diaziquone (NSC 182986) alone and in combination with other antineoplastic drugs to treat six human glioma and one human medulloblastoma tumor lines growing s.c. in athymic mice. Pharmacokinetic studies of diaziquone in the plasma of athymic mice indicated rapid clearance with a half-life of approximately 11.5 min. Diaziquone produced significant growth delays in at least one experiment using each of seven different tumor lines, and it produced consistent and significant delays in five of the seven. There was no obvious difference between a single dose and a dose administered once daily for 5 days, and tumor regressions to a volume smaller than that at treatment were uncommon in any of the single-drug experiments. Using our most extensively characterized human glioma line, D-54 MG, we found striking enhancement of the therapeutic effect by using nontoxic combinations of either diaziquone and carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, NSC 409962) or diaziquone and procarbazine (NSC 77213). These combinations produced significant increases in the median growth delay, significant increases in the number of tumor regressions, and some instances in which no palpable tumors were present 100 days after treatment. In contrast, in experiments using diaziquone -based chemotherapy combinations with either cyclophosphamide, cis-platinum, or vincristine, there was only slight enhancement of the therapeutic effect. These results, using human glioma and medulloblastoma tumor lines in athymic mice, suggest a broad range of activity of diaziquone against primary nervous system tumors and enhancement of its therapeutic effect with either 1,3-bis(2-chloroethyl)-1-nitrosourea or procarbazine. If Phase II and Phase III clinical trials corroborate these findings, the value of the nude mouse system for the evaluation of new therapeutic approaches to brain neoplasms would be further confirmed.

Arene imines, a new class of exceptionally potent mutagens in bacterial and mammalian cells.

K-region aziridines of polycyclic aromatic hydrocarbons reverted Salmonella typhimurium his- (TA100, TA98) and Escherichia coli trp- strains (WP2 uvrA), without requiring activation by mammalian enzymes. The number of revertants induced per nmol in S. typhimurium TA 100, the most responsive strain, variea from 6 to 10,000 for the seven monoaziridines and the two bisaziridines tested. Interestingly, the mutagenic potencies (y) of the monoaziridines were closely related (r = 0.984) with those of the corresponding epoxide analogues (x) by the equation y = 19.6 X0.97, i.e., the aziridines were about 20-fold stronger mutagens than were the epoxides. One of the aziridines, benzo(a)pyrene (BP)-4,5-imine, was investigated in several additional mutagenicity test systems: toxicity in DNA repair-deficient (rec-) and -proficient (rec+) Bacillus subtilis strains; induction of 6-thioguanine resistance in V79 Chinese hamster cells; and induction of sister chromatid exchanges in cultured human fibroblasts. In all systems, BP-4,5-imine was much more active than the epoxide analogue, BP-4,5-oxide. The difference in activity was particularly large in the two test systems with mammalian target cells in which several hundredfold higher concentrations of the epoxide had to be used in order to elicit equipotent effects. Even r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-BP, which is one of the most potent mutagens known for V79 cells, was less active in the mammalian cells than was BP-4,5-imine. One reason that arene imines are such potent mutagens may be that they are poorly detoxified. Addition of highly purified microsomal epoxide hydrolase, which strongly reduced the mutagenicity of BP-4,5-oxide and benz(a)anthracene-5,6-oxide in S. typhimurium, had no effect on the mutagenicity of the corresponding aziridines. Furthermore, while benz(a)anthracene-5,6-oxide was inactivated by highly purified cytosolic epoxide hydrolase, benz(a)anthracene-5,6-imine was not inactivated. It is noteworthy that the arene imines are isomeric with and structurally closely related to aromatic amines. Some aziridines derived from nonaromatic structures (ethylene imines) have been reported as metabolites of xenobiotics; others are used as chemotherapeutics. At present, however, the results are mainly of theoretical interest in that a new type of arene derivatives with exceptionally potent, probably ultimate, mutagenicity was discovered and may be exploited for the study of mechanisms of chemical carcinogenesis.

Toxicity of aziridinylbenzoquinone for aerobic and hypoxic cells of a transplanted mouse mammary tumor and interaction of the drug with radiation and adriamycin.

Aziridinylbenzoquinone (AZQ), an experimental drug with good tissue penetration, was tested against aerobic and hypoxic cells of the 16/C transplantable mouse mammary tumor. The drug was given alone or with local tumor radiation to kill most of the aerobic cells. The end point of response was growth delay. AZQ gave additive effects with tumor irradiation and was equally effective against aerobic and hypoxic cells. AZQ was also given in combination with Adriamycin, since the latter drug is known to have poor penetration in solid tissue. Combined treatment led to greater antitumor effects than were obtained with maximally tolerated doses of either drug alone. Isobologram analysis of the interaction between the drugs suggested a superadditive effect, and the addition of Adriamycin had little effect on myelosuppression induced by AZQ. The combination of AZQ and Adriamycin is worthy of further study.

Phase II and pharmacokinetic study of aziridinylbenzoquinone [2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone, diaziquone, NSC 182986] in high-grade gliomas.

2,5-Diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone (AZQ; Diaziquone, NSC 182986) is a rationally designed antitumor drug possessing sufficient lipid solubility to allow penetration into the central nervous system. Thirty-one patients with high-grade glioma and progressive disease following radiation, with or without previous chemotherapy, have been treated with 144 cycles of drug, consisting of 20 mg/sq m given as an i.v. infusion on Days 1 and 8 of a 28-day cycle. Responses were measured by serial computer tomography scanning. Of the 28 evaluable patients, 6 (21%) had limited improvement (10 to 40% reduction in tumor size) on computer tomography scan, 10 (36%) had disease stabilization, and 12 (43%) had progressive disease. The drug was well tolerated clinically, with little acute toxicity. The major toxicity was myelosuppression, which appeared cumulative, using this dose regimen. AZQ was measurable in both tumor tissue and tumor cyst fluid in patients on therapy. Plasma samples taken during the period of infusion confirm that 50% or more of the total AZQ exposure occurs during the infusion period. AZQ behaves as intended by design and demonstrates activity in this poor-prognosis group of patients.