Aberration induction by mitomycin C in early primary spermatocytes of mice

MC is well known to induce dominant lethal mutations in mouse spermatocytes. Tests were done to determine whether chromosomal aberrations could be identified in spermatocytes as being responsible for the dominant lethal effects. Male mice were treated with single doses of MC during DNA synthesis preceding meiosis and during early prophase of meiosis. Simultaneous labeling was performed to identify cells that were in S-phase during the time of treatment. Diakineses-metaphases I were analyzed for the occurrence of univalents, gaps, fragments and rearrangements. The frequencies of cells with aberrations increased with dose and time after treatment. Maximal values were obtained after 12 days, indicating that MC was most effective in cells undergoing DNA replication. 95% of these cells were labeled. The majority of aberrant cells contained one or more fragments. These cells will lead to dominant lethality of the zygotes after fertilization. Cells with rearrangements occurred 11 and 12 days after treatment. These cells can develop into sperm carrying a reciprocal translocation which would then give rise to semi-sterile progeny after fertilization. Further investigations are needed to study the transmission of rearrangements observed in primary spermatocytes.     

Inhibition of DNA cross-linking by mitomycin C by peroxidase-mediated oxidation of mitomycin C hydroquinone.

Mitomycin C requires reductive activation to cross-link DNA and express anticancer activity. Reduction of mitomycin C (40 microm) by sodium borohydride (200 microm) in 20 mm Tris-HCl, 1 mm EDTA at 37 degrees C, pH 7.4, gives a 50-60% yield of the reactive intermediate mitomycin C hydroquinone. The hydroquinone decays with first order kinetics or pseudo first order kinetics with a t(12) of approximately 15 s under these conditions. The cross-linking of T7 DNA in this system followed matching kinetics, with the conversion of mitomycin C hydroquinone to leuco-aziridinomitosene appearing to be the rate-determining step. Several peroxidases were found to oxidize mitomycin C hydroquinone to mitomycin C and to block DNA cross-linking to various degrees. Concentrations of the various peroxidases that largely blocked DNA cross-linking, regenerated 10-70% mitomycin C from the reduced material. Thus, significant quantities of products other than mitomycin C were produced by the peroxidase-mediated oxidation of mitomycin C hydroquinone or products derived therefrom. Variations in the sensitivity of cells to mitomycin C have been attributed to differing levels of activating enzymes, export pumps, and DNA repair. Mitomycin C hydroquinone-oxidizing enzymes give rise to a new mechanism by which oxic/hypoxic toxicity differentials and resistance can occur.     

The induction of hepatic cytochrome P-450 in C57 BL/10 and DBA/2 mice by isosafrole and piperonyl butoxide. A comparative study with other inducing agents

Styrene monooxygenase activity was measured in intact nuclear preparations from rat liver by means of a gas chromatographic method. Styrene epoxide formation is NADPH-dependent although it is enhanced when NADH is added with NADPH. This activity is inhibited by microsomal monooxygenase inhibitors SKF 525A and metyrapone and by microsomal epoxide hydrase inhibitors 1,2-epoxy-3,3,3-trichloropropene oxide and cyclohexene oxide. The percentage of inhibition is quantitatively dffferent for the four compounds. Known inducers of liver microsomal monooxygenase show different patterns of induction on nuclear preparations. Phenobarbital induces nuclear monooxygenase activity more than the respective microsomal activity, whereas the contrary holds true for β-naphthoflavone.     

Bioreductive activation of mitomycin C by DT-diaphorase.

The role of DT-diaphorase (DTD, EC 1.6.99.2) in the bioreductive activation of mitomycin C was examined using purified rat hepatic DTD. The formation of adducts with reduced glutathione (GSH), binding of [3H]mitomycin C to DNA, and mitomycin C-induced DNA interstrand cross-linking were used as indicators of bioactivation. Mitomycin C was metabolized by DTD in a pH-dependent manner with increasing amounts of metabolism observed as the pH was decreased from 7.8 to 5.8. The major metabolite observed during DTD-mediated reduction of mitomycin C was 2,7-diaminomitosene. GSH adduct formation, binding of [3H]mitomycin C and mitomycin C-induced DNA interstrand cross-linking were observed during DTD-mediated metabolism. In agreement with the pH dependence of metabolism, increased bioactivation was observed at lower pH values. Temporal studies and experiments using authentic material showed that 2,7-diaminomitosene could be further metabolized by DTD resulting in the formation of mitosene adducts with GSH. DNA cross-linking during either chemical (sodium borohydride) or enzymatic (DTD) mediated reduction of mitomycin C could be observed at pH 7.4, but it increased as the pH was decreased to 5.8, showing the critical role of pH in the cross-linking process. These data provide unequivocal evidence that the obligate two-electron reductase DTD can bioactivate mitomycin C to reactive species which can form adducts with GSH and DNA and induce DNA cross-linking. The use of mitomycin C may be a viable approach to the therapy of tumors high in DTD activity, particularly when combined with strategies to lower tumor pH.     

Sequence-specific DNA damage induced by reduced mitomycin C and 7-N-(p-hydroxyphenyl)mitomycin C.

Mitomycin C reduced with sodium borohydride induced the DNA damage at deoxyguanosines preferentially in dinucleotide sequence G-T. The DNA damage produced strand breaks when subsequently heated. The DNA damage scarcely occurred when the end-labeled DNA was preincubated with ethidium bromide or actinomycin D before the addition of mitomycin C and the reducing agent. Fully reduced mitomycin C did not induce the DNA damage. The mitomycin C-inducing DNA damage seems to require the intercalation of the partially reduced mitomycin C of short life time, probably semiquinone radical, between DNA base pairs. The inhibitory effects of sodium chloride and radical scavengers suggested that the requirement of the covalent bond formation of mitomycin C to DNA and the involvement of oxygen radicals in the DNA damage. 7-N-(p-hydroxyphenyl)mitomycin C, which is reported to show a higher antitumor activity and a lower toxicity than mitomycin C, was readily reduced with dithiothreitol and induced the sequence-specific DNA damage, whereas mitomycin C was not.     

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac⁻) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types—those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac⁺ transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.     

Comparative inducibility of bacteriophage in naturally lysogenic and lysogenized strains of Listeria spp. by u.v. light and Mitomycin C

A total of 34 listeria lysogens, which are either natural phage carriers or artificially lysogenized strains, were tested for production of prophage upon treatment with u.v. light and Mitomycin C (MC). Most strains were readily inducible, in particular the generated prophage carriers tested. The yield of free phage could be increased up to 1600-fold. With respect to the listeria lysogens investigated, MC was found to be a more powerful inducing agent than u.v. light. Thus, it should be further applied in screening for new phages.     

Changes in protein synthesis on mitomycin C induction of wild-type and mutant CloDF13 plasmids.

Mitomycin C treatment of Escherichia coli K-12 cells containing the nonconjugative plasmid CloDF13 resulted in inhibition of host chromosome protein synthesis and a high rate of synthesis of two CloDF13-specified proteins whose molecular weights correspond to cloacin and immunity protein. Five molecules of immunity protein were synthesized for each cloacin DF13 molecule. Mitomycin C-treated cells containing a copy mutant of CloDF13 made three to four times as much of each protein as cells containing wild-type CloDF13. CloDF13 plasmids that contained the transposon Tn1 were isolated. Two did not induce after mitomycin C treatment, failing both to inhibit host cell synthesis and to produce the two new proteins. In minicells, they showed reduced CloDF13-specified protein synthesis and produced three Tn1-specified proteins.     

Towards an improved micronucleus test Studies on 3 model agents,mitomycin C,cyclophosphamide and dimethylbenzanthracene

Although the micronucleus assay has been widely used to detect chromosomal breakage in vivo, none of the protocols that have been used have been fully justified either experimentally or theoretically. Accordingly, we have studied the production of micronuclei by 3 compounds in detail, hoping that an optimal protocol could be defined. 2 factors were investigated, the time course of micronucleus production and the influence of multiple injections. The time course of micronucleus production in polychromatic erythrocytes (PCE) proved to be different for each of the chemicals studied, cyclophosphamide, mitomycin C and dimethylbenzanthracene. After a single intraperitoneal injection, for example, the maximum frequency of micronuclei occurred at ∼36 h for mitomycin C, and at ∼72 h for dimethylbenzanthracene. In no case could an increase in micronuclei be detected earlier than 10 h after injection, as would be predicted on theoretical grounds. From this we conclude, firstly, that multiple times are more valuable than multiple doses in detecting chromosomal breakage in vivo, and, secondly, that any treatment within ∼10 h of sampling is ineffective.The effect of multiple injections was also investigated directly. The conclusion that injections within 10 h of sampling are ineffective was confirmed. In general, the effect of 2 injections 24 h apart was close to the sum of the effects that the individual injections produce at the corresponding times. Obviously if this additivity is the usual situation, and more chemical can be given in multiple injections than in a single one, the sensitivity of the assay will be improved by the use of an appropriate multi-injection protocol. Multiple injections, however, were found to have subtractive effects if carried too far. Although we do not believe that we can define the most effective protocol yet, our results suggest that the following protocol should be more successful than those used previously: (1) inject intraperitoneally at 0 and 24 h, sample at 48, 72 and 96 h, (2) if a negative result is obtained, inject intraperitoneally at 0 h and sample at 30, 48 and 72 h. In each case, a positive result can be confirmed by either a repeat experiment or a dose—response study at the time of the maximum response.     

Induction of cytoplasmic petite in yeast by guanidine hydrochloride: Combined treatment with other inducing agents

We have studied the induction of ?^? mutants by guanidine hydrochloride (GuHCl) in combination with other known inducers: ethidium bromide (EB), berenil and ultraviolet light. Competition was observed when cells were simultaneously treated with optimal concentrations of EB and GuHCl; on the other hand, treatment of cells with EB in the presence of non-inducing concentrations of GuHCl resulted in the stimulation of ?^? induction by EB. Furthermore, using a strain which upon treatment with high EB concentrations shows recovery of respiratory competence, the presence of GuHCl did not interfere either with the early phase of induction or with the recovery phase, but it did interfere in a competitive fashion with the final irreversible phase of EB induction. In the case of berenil, a synergistic effect was seen when cells were pretreated with GuHCl. A synergistic induction was also observed when cells were submitted to UV prior to GuHCl treatment. These results suggest that GuHCl, EB and berenil act via some common step in their ?^? induction pathways. Moreover, GuHCl may somehow be decreasing the efficiency of dark repair of ultraviolet lesions on mitochondrial DNA.     

Mycobacterial helicase Lhr abets resistance to DNA crosslinking agents mitomycin C and cisplatin

Mycobacterium smegmatis Lhr exemplifies a novel clade of helicases composed of an N-terminal ATPase/helicase domain (Lhr-Core) and a large C-terminal domain (Lhr-CTD) that nucleates a unique homo-tetrameric quaternary structure. Expression of Lhr, and its operonic neighbor Nei2, is induced in mycobacteria exposed to mitomycin C (MMC). Here we report that lhr deletion sensitizes M. smegmatis to killing by DNA crosslinkers MMC and cisplatin but not to killing by monoadduct-forming alkylating agent methyl methanesulfonate or UV irradiation. Testing complementation of MMC and cisplatin sensitivity by expression of Lhr mutants in Δlhr cells established that: (i) Lhr-CTD is essential for DNA repair activity, such that Lhr-Core does not suffice; (ii) ATPase-defective mutant D170A/E171A fails to complement; (iii) ATPase-active, helicase-defective mutant W597A fails to complement and (iv) alanine mutations at the CTD–CTD interface that interdict homo-tetramer formation result in failure to complement. Our results instate Lhr''s ATP-driven motor as an agent of inter-strand crosslink repair in vivo, contingent on Lhr''s tetrameric quaternary structure. We characterize M. smegmatis Nei2 as a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Counter to previous reports, we find Nei2 is inactive as a lyase at a THF abasic site and has feeble uracil glycosylase activity.