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The anticancer drug mitomycin C produces cytotoxic effects after being converted to a highly reactive bis-electrophile by a reductive activation, a reaction that a number of 1-electron or 2-electron oxidoreductase enzymes can perform in cells. Several reports in the literature indicate that ascorbic acid can modulate the cytotoxic effects of mitomycin C, either potentiating or inhibiting its effects. As ascorbic acid is a reducing agent that is known to be able to reduce quinones, it could be possible that the observed modulatory effects are a consequence of a direct redox reduction between mitomycin C and ascorbate. To determine if this is the case, the reaction between mitomycin C and ascorbate was studied using UV/Vis spectroscopy and LC/MS. We also studied the reaction of ascorbate with mitomycin A, a highly toxic member of the mitomycin family with a higher redox potential than mitomycin C. We found that ascorbate is capable to reduce mitomycin A efficiently, but it reduces mitomycin C rather inefficiently. The mechanisms of activation have been elucidated based on the kinetics of the reduction and on the analysis of the mitosene derivatives formed after the reaction. We found that the activation occurs by the interplay of three different mechanisms that contribute differently, depending on the pH of the reaction. As the reduction of mitomycin C by ascorbate is rather inefficiently at physiologically relevant pH values we conclude that the modulatory effect of ascorbate on the cytotoxicity of mitomycin C is not the result of a direct redox reaction and therefore this modulation must be the consequence of other biochemical mechanisms., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53-independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-beta-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC. We find the increase in mitosene 1-beta-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-beta stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein.

Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other than a selective defect in cross-link repair.

The luminous intensity of dark variant (S1) separated from photobacterium phosphoreum (A2) was 1/10,000 less than that of wild-type. Ethidium bromide (EB) (0.6 mg/L), Mytomycin C (MC, 0.05 mg/L), 2-amino fluorene (2-AF, 1.0 mg/L) all could strongly induce reversion mutation for S1 within 24 h and increase reversion ratio significantly. The results of experiments indicated that these revertants had stable genetic characteristic and the mutation may take place at gene levels. The mutagenesis to S1 caused by EB, MC and 2-AF was detected and it may be used as a new rapid, simple and sensitive method for gene toxicant monitoring.

An experimental model for pulmonary toxicity of KW-2149, a new mitomycin C analogue, was established and the inhibitory effects of dexamethasone (DM) were investigated. KW-2149 was given to male rats 3 or 5 times at weekly intervals by intravenous injection of 3.28 or 8.2 mg/kg. As a suitable model for pulmonary toxicity, the dose of 3.28 mg/kg per week for 3 weeks was selected, this causing exudative pleural effusion in all animals but no deaths. For preventing this toxicity, DM was injected subcutaneously 3 times every week at 0.5, 1.0, 2.5 or 5.0 mg/kg. The 0.5 mg/kg dose was sufficient to completely prevent development of pleural effusions. Combined DM treatment may be an effective chemotherapy for KW-2149 induced pulmonary toxicity.

Background: Chemoresistance of tumor cells is involved with many factors, one of which is the P-glycoprotein function to pump anthracyclines out of cells. 99mTc-MIBI accumulates in several tumors, and some of these cells wash out 99mTc-MIBI through P-glycoprotein., Materials and Methods: We investigated if the wash-out of 99mTc-MIBI from the tumor in fifteen female patients with breast cancer could be related with the chemosensitivity of anticancer agents; doxorubicin (DOX), epirubicin (FAM), pinorubicin (PINO), mitomycin (MMC), cisplatin (CDDP), and 5-fluorouracil (5-FU), in each tumor tissues. The wash-out of 99mTc-MIBI, defined as retention index, was quantified from an early and delayed 99mTc-MIBI imaging. The chemosensitivity of the anticancer agent, and inhibition ratio, was determined in vitro assay by using surgical specimens obtained from patients who underwent 99mTc-MIBI imaging. P-glycoprotein in the surgical specimen was studied by immunohistochemical staining on its paraffin section using a monoclonal antibody., Results: Inhibition ratio of anthracycline agent, DOX, FAM or PINO, was well correlated with retention index of 99mTc-MIBI with coefficient of 0.75, 0.60, or 0.62, respectively, whereas a poor relationship was observed for MMC and CDDP. The retention indices of 99mTc-MIBI were remarkably small for patients in the P-glycoprotein positive group., Conclusion: 99mTc-MIBI retention index quantified from its early and delayed scintigraphy is a good indicator to predict the chemosensitivity of anthracyclines in untreated breast cancer.

Pulmonary side effects are increasingly observed as dose-limiting toxicity (DLT) of cancer treatment. The available preclinical models have a limited predictive value for lung toxicity in humans. We have attempted to elucidate potential mechanisms involved in these reactions, by studying the effects on cells, possibly involved in these reactions after in vitro exposure to drugs with known lung toxic effects. We have investigated the effects of bleomycin (BLM), mitomycin C (MMC), KW-2149 and its two known metabolites, M16 and M18, on oxygen radical production by granulocytes, on cytokine production: interleukin (IL)-6, transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha by a human macrophage cell line (THP-1), by human endothelial cells (HVEC and HMEC) and a human colorectal cancer cell line (DLD-1), and on the cytotoxicity on endothelial cells in both confluent and non-confluent culture. The generation of oxygen radicals by normal and pre-stimulated granulocytes was not increased after preincubation with any of the drugs, at the concentrations tested. None of the cytokines (IL-6, TNF-alpha or TGF-beta) was found significantly increased in culture medium after exposure to any of the mitomycins. This was in contrast with the effect of BLM incubation, causing a rise in TGF-beta concentration. Both types of endothelial cells showed a dose-dependent, exposure duration-dependent, proliferation inhibition for all agents tested. This inhibitory effect was clearly proliferation dependent as shown by the increased inhibition in semi-confluent as opposed to confluent endothelial cell cultures. Both mitomycins tested were more cytotoxic than BLM to both confluent and proliferating endothelial cells.

Overexpression of the trans-membrane drug efflux pump P-glycoprotein is one of the major mechanisms by which cancer cells develop multidrug resistance. We demonstrated previously that noncytotoxic doses of various genotoxic chemicals, particularly DNA cross-linking agents, preferentially altered expression of inducible genes. These effects occurred principally at the transcriptional level and were closely correlated temporally with DNA damage. Because the mdr1 gene coding for P-glycoprotein has been reported to be highly inducible, we were interested in the effects of genotoxic cancer chemotherapy agents on its expression. We report that the DNA cross-linking agent mitomycin C significantly suppressed mRNA and protein expression of P-glycoprotein and decreased the rate of drug efflux. Mitomycin C pretreatment also significantly increased the sensitivity of cancer cells to subsequent killing by the P-glycoprotein substrate doxorubicin, decreasing the ED50 by 5- to 10-fold. Suppression of P-glycoprotein expression was also observed with subtoxic doses of the DNA cross-linking agents cisplatin, BMS181174, and chromium(VI). These effects occurred in both human and rodent cell lines; in cell lines derived from colon, breast, leukemia, neuroblastoma, and hepatoma tumors; and under both monolayer and "spheroid" culture conditions. These results suggest the basis for novel clinical cancer chemotherapy regimens aimed at drug-resistant tumors, in which a sub-chemotherapeutic dose of a DNA cross-linking agent is used to modulate the multidrug resistance phenotype prior to treatment with a second cytotoxic agent.

The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (E. O. Davis, S. G. Sedgwick, and M. J. Colston, J. Bacteriol. 173:5653-5662, 1991). In this study, the expression of this gene was shown to be inducible in response to various DNA-damaging agents by using a transcriptional fusion to the reporter gene encoding chloramphenicol acetyltransferase. A segment of DNA around 300 bp upstream of the coding region was shown to be required for expression. However, primer extension analysis indicated that the transcriptional start sites were 47 and 93 bp upstream of the translation initiation codon. Sequence motifs with homology to two families of Escherichia coli promoters but also with significant differences were located near these proposed transcription start sites. The differences from the E. coli consensus patterns would explain the previously described lack of expression of the M. tuberculosis recA gene from its own promoter in E. coli. In addition, the M. tuberculosis LexA protein was shown to bind specifically to a sequence, GAAC-N4-GTTC, overlapping one of these putative promoters and homologous to the Bacillus subtilis Cheo box involved in the regulation of SOS genes. The region of DNA 300 bp upstream of the recA gene was shown not to contain a promoter, suggesting that it functions as an upstream activator sequence.

Three carbamate insecticides (propoxur, methomyl, and aldicarb) were evaluated for their ability to induce micronuclei (MN) in vitro using cultured Chinese hamster ovary (CHO) cells, and in vivo in mouse bone marrow erythrocytes. In vitro, all three insecticides induced a significant increase in micronucleated binucleate cells, which was generally both dose and sample time dependent. The in vivo studies involved treating male BALB/c mice by different routes, either once or on 3 consecutive days, followed by multiple or single sampling. Treatment by intraperitoneal injection or oral gavage induced a significant increase in micronucleated reticulocytes (MNRETs) in peripheral blood. For all three chemicals, the MN response depended on sample time and the number of treatments, while for aldicarb, the response depended also on the route of exposure. These positive results demonstrate that propoxur, methomyl, and aldicarb are capable of inducing structural and/or numerical chromosomal aberrations in mammalian cells either in vitro or in vivo. Furthermore, based on the results obtained, on optimal in vivo MN protocol for carbamate insecticides is a single treatment followed by blood sampling at 24 and 48 hr after treatment.

The absolute configuration at the C-1 position of a 1,10-bisacetoxymitosene (WV15) appears to be important for enzymatic reduction, DNA interstrand cross-linking and in vitro antitumour activity of this compound. DNA cross-linking by the (-)-(S)-enantiomer of WV15 upon reduction with sodium dithionite (Na2S2O4) was more efficient than cross-linking by the (+)-(R)-enantiomer. Also, following enzymatic two-electron reduction by DT-diaphorase or one-electron reduction by xanthine oxidase, (-)-(S)-WV15 was more efficient in DNA cross-linking than (+)-(R)-WV15. However, the difference in cross-linking efficiency was less than upon chemical reduction, and in the case of enzymatic reduction that higher amount of DNA cross-links formed by (-)-(S)-WV15 can be explained by more efficient enzymatic activation of this enantiomer as compared to (+)-(R)-WV15. The enantiomeric preference upon chemical reduction can be explained by a second chemical reduction of DNA-bound WV15, which presumably does not occur upon enzymatic reduction. (-)-(S)-WV15 appeared to be more active than its (+)-(R) counterpart in A204 and L1210 tumour cell lines, with (+)-(R)/(-)-(S) toxicity ratios as high as 200 and 68, respectively. In Chinese hamster V79 cell lines, toxicity of the enantiomers was measured under oxic and hypoxic conditions. The oxic/hypoxic toxicity ratios of (+)-(R)-and (-)-(S)-WV15 in the Chinese hamster V79 cell line were 5.5 and 2.4, respectively. These different oxic/hypoxic toxicity ratios may indicate that different reducing enzymes are involved in the activation of the enantiomers. Generally, in biological systems, different activities of (+)-(R)- and (-)-(S)-WV15 appear not to be caused by different intrinsic cross-linking capacities of the enantiomers, but by more efficient enzymatic activation of (-)-(S)-WV15, as compared to (+)-(R)-WV15. The (-)-(S)-enantiomer of WV15 appears to be more active both in in vitro tumour models and in DNA cross-linking assays, and therefore the absolute configuration of mitosenes is indicated to be important for the antitumour activity of these compounds.

The ability of melatonin to influence lipopolysaccharide (LPS)-induced genotoxicity was tested using micronuclei as an index in both bone marrow and peripheral blood cells of rats. LPS was given as a single dose of 10 mg/kg. Melatonin (5 mg/kg) was injected prior to LPS administration and thereafter at 6 h intervals to the conclusion of the study (72 h). The number of micronucleated polychromatic erythrocytes increased significantly after LPS administration both in cells from peripheral blood and bone marrow. Melatonin administration to LPS-treated rats highly significantly reduced micronuclei formation in both peripheral blood and bone marrow cells beginning at 24 h after LPS administration and continuing to the end of the study. In blood the increase in micronuclei formation was time-dependent in LPS-treated rats with peak values being reached at 36-48 h. The ability of melatonin to reduce LPS-related genotoxicity is likely related to its antioxidant activity.

This investigation was aimed at determining the possible relationship between DNA interstrand cross-linking and the cytotoxic activity of potential antitumour mitosene compounds. Mitosenes, possessing two good leaving groups at C-1 and C-10, were found to be able to cross-link calf thymus DNA under hypoxic conditions following sodium dithionite (Na2S2O4) reduction at pH 7.0 and pH 5.5. DNA interstrand cross-linking was pH dependent for most of the mitosenes used, with a higher amount of cross-links formed at pH 5.5 compared to pH 7.0. Without reduction or under aerobic conditions no cross-link formation was detected. The importance of DNA damage for the toxic effect of these mitosenes was assayed by comparing the survival in a DNA repair deficient and a DNA repair proficient E. coli K-12 strain. A correlation between the number of cross-links formed in calf thymus DNA in vitro and the IC50 values in the DNA repair deficient E. coli strain was found. The effect of hypoxia on toxicity of mitosenes was studied in Chinese hamster V79 cells. In these cells, mitosenes appeared to be very active. Under severe hypoxic conditions toxicity of these mitosenes increased, most likely due to the increased lifetime of the activated mitosene species as compared to aerobic conditions. The results suggest that DNA cross-linking following reductive activation is important for the eventual activity of mitosenes in a bacterial system. Increased activity of mitosenes under hypoxic conditions in the V79 cells indicates that these mitosenes may be more active in hypoxic parts of tumours.

Molecular orbital calculations based on coordinates from X-ray analysis have been performed for a set of 24 mitomycins, of which eight compounds have not been isolated so far. To prioritize the order of synthesis of these missing compounds, a new method named DISCA (distance and charge analysis) has been developed. DISCA screens correlations between spatial distribution of charge in molecules and their biological activity. The spatial distribution of charge is represented by several indexes in DISCA. LD50 and ED50 were used as measures of biological activity. DISCA has successfully extracted indexes which have significantly high correlation coefficients. The indexes with the highest correlation coefficient were common to both LD50 and ED50. By use of the correlation functions with high correlation coefficients DISCA has predicted that 9-epi-1a-N-demethylmitomycin D should have the best ED50 and a modest LD50 among the missing mitomycins.

One hundred and ten patients with primary epidermoid cancers of the anal canal were treated in a series of prospectively designed, nonrandomized protocols of split-course radiation therapy with concurrent administration of 5-fluorouracil (5-FU) with or without mitomycin. The addition of mitomycin was associated with improved primary tumor control rates (87 vs. 58% at 4 years, p = 0.005) and improved 4-year actuarial cause-specific survival (80 vs. 64%, p = 0.02). Hematologic toxicity was the most frequent acute side effect of mitomycin use. No long-term toxicity was attributed to mitomycin only. Mitomycin appeared to benefit patients principally through improved control of cancer in the irradiated volume; there was no evidence of reduced risk of extrapelvic metastases. Several investigators have reported high rates of control of epidermoid anal cancers with preservation of anorectal function following concurrent treatment with mitomycin, 5-FU, and radiation. Mitomycin's role in anal cancer is being evaluated in a randomized clinical trial by the Radiation Therapy Oncology Group. The mechanisms of any interactions between mitomycin and radiation or other cytotoxic drugs in clinical practice remain to be determined.

Mitomycin, ifosfamide, and cisplatin have demonstrated the best single-agent activity thus far in patients with non-small cell lung cancer (NSCLC), the most common malignant disease in the western world. For this reason, we initiated a phase II study, giving these three agents in combination (designated MIC) to 74 patients with inoperable NSCLC. Sixty-six patients were evaluable for response, of whom 30 (45%) demonstrated a partial response and 7 (11%) a complete response. These results, along with those obtained in two other phase II trials of MIC in NSCLC, promoted us to begin a large-scale, multicenter, phase III study of MIC in patients with inoperable limited-stage NSCLC. In this ongoing study, patients have been randomized to receive treatment with MIC and radiotherapy or radiotherapy alone. We hope to resolve the issue of whether a survival advantage is conferred on NSCLC patients treated with radiotherapy in combination with this promising chemotherapeutic regimen.

Mitomycin has proven to be among the most active drugs available for the single-agent treatment of non-small cell lung cancer (NSCLC). In combination with vinca alkaloids and cisplatin, mitomycin can produce response rates greater than or equal to 50% in properly selected patients. In our experience, such responses were achieved using moderate doses (7 or 8 mg/m2) of mitomycin, which also resulted in fewer hematologic and other toxicities. Delivery of MVP (mitomycin/vinca alkaloid/cisplatin) to 150 patients with stages III and IV NSCLC during the last decade showed maximal response was achieved after two or three cycles of therapy. A comparative analysis of results reported using MVP regimens suggests that high response rates are associated with greater dose-intensive use of cisplatin and lesser dose-intensive use of mitomycin. Although the role of MVP in the treatment of advanced NSCLC is unclear, use of mitomycin-containing regimens as part of a multidisciplinary approach to stage IIIA NSCLC has yielded high response rates and has successfully downstaged patients prior to surgery. Randomized clinical trials will be required to validate these findings, but the focus of future research should be on discovering new agents with greater activity and on developing new approaches wherein these agents can be delivered with maximum efficacy and minimum toxicity.

Chromosomal aberration (CA) and sister-chromatid exchange (SCE) frequencies have been assessed in 9 patients with von Recklinghausen's neurofibromatosis (NF1) and 8 apparently healthy controls. In separate experiments over a 5-year period, blood lymphocytes, skin fibroblast cell strains, and lymphoblastoid lines from both groups were treated with X-rays or mitomycin C (MMC) to determine whether the NF1 group was more sensitive to these agents than the control group. No difference between cells from NF1 patients and controls was observed with respect to spontaneous or X-ray-induced CA. Spontaneous or X-ray- and MMC-induced SCE frequencies were also similar in NF1 patients and controls.

Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.

The alkylating antitumor agents mitomycin A (MMA), mitomycin C (MMC), and seven N7 analogs were compared in terms of their cardiotoxic and antitumor activity in vitro. Neonatal rat-heart myocytes were sensitive to five of the compounds studied, including MMA, 7-dimethylamidinomitosane (BMY-25282), 7-(N-methyl-piperazinyl)-mitosane (RR-194), N7-(4-iodophenyl)-MMC (RR-208), and N7-(4-hydroxyphenyl)-MMC (M-83) in order of descending molar potency. MMA and RR-208 possessed the greatest cytotoxic potency against 8226 human myeloma tumor cells in vitro. Two of the nine mitomycins studied, BMY-25282 and M-83, showed greater cytotoxic potency for heart cells. For these two agents, the ratio of the 50% inhibitory concentration in heart cells to that in 8226 myeloma cells was 50 and 32, respectively. For the other analogs, the tumor-cell cytotoxic potency was much higher (ranging from 200 to 7,000). For the nine mitomycin compounds, a correlation was found between heart-cell toxicity and low reduction potentials (E1/2 values) ranging from -0.16 to -0.37 V. Thus, as the reduction potential decreased (easier reducibility), the cardiotoxic potency in vitro increased (r = 0.81). In contrast, mitomycins with reduction potentials of higher than -0.37 V were much less potent cardiotoxins. Thus, mitomycin C (E1/2 = -0.45 V) was noncardiotoxic even when tested at concentrations 100-fold above those pharmacologically achievable in humans. Mitomycin C also failed to enhance doxorubicin (Adriamycin) cardiotoxicity in vitro. Importantly, no correlation was found between the reduction potential and the antitumor activity of the nine analogs (n = 0.51), in this small series.

The induction of genetic damage has clear and dramatic implications for human health, with teratogenic, mutagenic, cataractogenic and carcinogenic consequences resulting from cellular chromosomal alterations in appropriate tissues. When analysing the potential of an agent to initiate genetic damage or in evaluating possible incumbent genomic damage a variety of complementary assays may be employed. These apply to cells in vitro, to in vivo assessments involving small mammals and most importantly to derived human cells and tissues including those of ocular origin. Cytogenetic assays have the important advantage that they enumerate damage at the level of the individual cell. Assays involving the examination of chromosomal aberrations at mitosis, of cells prior to mitosis using the technique of premature chromosome condensation, of micronuclei in post-mitotic cells and of sister chromatid exchanges will be described. The development of human chromosome specific probes and fluorescent in situ hybridisation (FISH) techniques combine the resolution of molecular biology with classical cytogenetics in a powerful approach to defining genomic change and its consequences. These techniques and assays can be further augmented by in situ cytometry such that overall a number of parameters can be quantified involving cellular kinetics, clastogen and/or aneugen definition and ultimately the establishment of dose response relationships. A rational basis for avoidance or control, for intervention or for defining probable cause of the role of genotoxicants in the development of human disease can then be established.

SOS mutagenesis in prokaryotes is dependent upon the inducible activity of the chromosomally encoded UmuDC proteins, or homologous proteins such as MucAB or ImpCAB which are found on naturally occurring plasmids. Relative to Escherichia coli, however, Salmonella typhimurium is much less responsive to the mutagenic effects of DNA-damaging agents, despite the fact that it possesses both chromosomally and plasmid encoded umu-like operons. In E. coli, activation of the UmuD mutagenesis protein to UmuD' via RecA-mediated proteolysis is a critical step in the mutation fixation pathway. We have used a polyclonal antiserum raised against the E. coli UmuD and UmuD' proteins to show that S. typhimurium expresses cross-reacting material only after treatment with the DNA-damaging agent mitomycin C. The S. typhimurium umuDC operon, therefore, appears to be regulated by mechanisms similar to the E. coli umuDC operon. After induction, the S. typhimurium UmuD protein was processed to UmuD' in both S. typhimurium and E. coli. However, the S. typhimurium UmuD protein appears to be cleaved more efficiently than the E. coli UmuD protein under similar conditions. The data suggest that conversion of UmuD to the mutagenically active UmuD' is not the rate-limiting factor accounting for the weakly mutable phenotype of S. typhimurium.

Most chemicals are S-dependent and are potent inducers of SCE, but do not produce chromosome-type aberrations in the first metaphases after exposure. Ionizing radiation, which is an S-independent agent, produces chromosome-type aberrations, especially dicentrics and rings, but inefficiently produces chromatid-type aberrations. A series of experiments has been performed to investigate whether cytogenetic damage induced by ionizing radiation (gamma-rays) might be assessed separately from that induced by the alkylating chemical, mitomycin C (MMC), when human lymphocytes were exposed to these 2 agents in combination. Whole-blood cultures of human lymphocytes in G0 phase were exposed to gamma-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA), analyzed in cultures incubated for 56 h without BrdUrd, and sister-chromatid exchanges (SCEs) in cultures incubated for 72 h with BrdUrd. The frequency of chromosome-type aberrations (dicentrics and rings) increased with increasing doses of gamma-rays from 0.5 to 4.0 Gy. The dose-response relationships were the same with or without concomitant treatment with MMC (10(-6) M). Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and gamma-rays (2 Gy). There was no interaction between MMC and gamma-rays concerning these 2 endpoints.

The comutagenic activity of selenium was investigated using in vitro and in vivo techniques, including the liquid suspension modification of the standard Salmonella/microsome mutagenicity assay, the metaphase analysis of chromosome aberrations in CHO cells and in mouse bone marrow as well as the micronucleus assay in mouse bone marrow. 4 h growth of S. typhimurium TA1535 in a nutrient broth containing 2.9 x 10(-5) M but not 1.16 x 10(-5) M Na2SeO3 caused an up to 10-fold increase of the number of N-methylnitrosourea (MNU, 2.0-2.5 mM)-induced his+ revertants and an up to 2-fold elevation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1.48 x 10(-5))-induced mutation rate. Pretreatment of bacteria with Na2SeO3 alone had no effect on the spontaneous mutation level. The combined treatment of CHO cells with MNNG (1.25 x 10(-5) M) or tobacco smoke (TS, 2-3 puffs generated by a cigarette inhalation machine) plus Na2SeO3 (0.58-1.16 x 10(-5) M) starting 2 h and 4 h before the MNNG or TS treatment respectively resulted in a 2-3-fold increase in the percent of metaphases with chromosome aberrations. Furthermore, treatment for 7-14 days of male BDF1 (C57Bl x DBA2) or CC57W mice with Na2SeO3, added to the drinking water at a concentration of 10 ppm, potentiated by 2-3 times the chromosome-damaging activity of urethane (0.5-1.0 g/kg, i.p.) in mouse bone marrow, as measured by the formation of micronuclei or chromosome aberrations. In addition, Na2SeO3 increased up to 43.8% the number of micronucleated polychromatic erythrocytes (MNPCE) induced by mitomycin C (MMC, 1.5 mg/kg, i.p.) in BDF1 mouse bone marrow. Treatment of mice with Na2SeO3 alone had no effect on the spontaneous level of MNPCE. All these findings are consistent with a comutagenic and coclastogenic activity of selenium both in prokaryotes and in eukaryotes, in vitro as well as in vivo after pretreatment of target cells with the trace element.

Menadione-resistant subclones were selected from cultured Chinese hamster lung (CHL) cells which had been mutagenized with MNNG or ENU. The frequency of surviving colonies and the level of resistance were higher in mutagenized cells than in non-mutagenized cells. A subclone (designated MM1) was isolated from MNNG-treated cells and showed the highest level of resistance, 3 times higher than the parental CHL cells. The level of resistance was stable in non-selective medium over 3 months. The MM1 cells were also 2-3 times more resistant to other naphthoquinones. The activity of NADPH-cytochrome P-450 reductase, which is thought to play an important role in activation of menadione, was reduced in the MM1 cells to half that in the parental CHL cells. On the other hand, no differences between MM1 and CHL cells were found in the activity of superoxide dismutase and catalase which are assumed to defend against the cytotoxicity of menadione. Karyotype analyses indicated that one small chromosome was lost in the MM1 cells. The MM1 cells showed a 3-fold resistance to menadione in the chromosomal aberration test. The frequencies of chromosomal aberrations induced by adriamycin and mitomycin C which could be activated by NADPH-cytochrome P-450 reductase were almost the same in the MM1 and CHL cells, suggesting that the reductive activation of these compounds by this enzyme in microsomes may not be involved in the induction of chromosomal aberrations.

Micronuclei (MN) in erythrocytes of mouse bone marrow cells were induced in vivo by the spindle poisons colchicine (COL) and vinblastine (VBL), by hydroquinone (HQ) and by the alkylating agent mitomycin C (MMC). Two different methods were applied to detect whole chromosomes with centromeric proteins or chromatin in MN to discriminate between spindle damaging or clastogenic activity of these chemicals. One method determined the fraction of MN with centromeric chromatin by immunofluorescent staining using antikinetochore antibodies (CREST staining). The other method applied non-radioactive in situ hybridization with a novel DNA probe. The fractions of MN that showed positive signals by either technique thus indicating with a high probability the presence of whole chromosomes instead of acentric fragments, were in good agreement for COL, VBL and HQ. After application of MMC, however, 4.5% of the MN were CREST-positive, while 29% gave a positive hybridization signal. The results suggest, that kinetochores may have lost certain centromeric antigens due to treatment with MMC so that MN containing whole chromosomes appear CREST-negative. The presented in situ hybridization scheme using satellite DNA is a more direct detection and is advantageous to the CREST staining technique in that it is unaffected by damage of kinetochore or centromeric function.

The effect of multiple treatment with chemicals in the micronucleus test was evaluated by simulation involving an estimation of the additive accumulation of micronucleated polychromatic erythrocytes (MNPCEs) on the basis of time-response data available on single treatments with mitomycin C, 1-beta-D-arabinofuranosylcytosine, 6-mercaptopurine, and methotrexate. The frequency of MNPCEs calculated for different multiple treatment regimens by the model could predict the effects observed in real experiments. On the other hand, the effect of multiple treatments on bone marrow depression, expressed as a decrease in the frequency of polychromatic erythrocytes, was exponential according to both the simulation and actual data. These results suggest that although increasing the number of treatments may additively enhance the MNPCE response obtained with some agents it may, in the case of bone marrow-toxic chemicals and doses, make micronucleus analysis more time-consuming and even impossible due to the exponential decrease of analyzable cells, especially in the case of manual scoring.

The effect of estrogen on the induction of micronucleated polychromatic erythrocytes (MPCE) by mutagens was examined in male mice. In the dose-response study, a dose-related inhibition of the mitomycin C (MMC)-induced MPCE frequency by estradiol (E2) treatment was observed. In the time study, inhibitory effects of E2 on MPCE frequency by MMC were observed when MMC was administered at 0 or 1 day after injection of E2. The most effective protocol for inhibition was when E2 and MMC were used on the same day. Of mutagens other than MMC, only vincristine (VCR) showed a significant decrease in MPCE frequency when used together with E2. Benzo[a]pyrene (BaP) and 5-fluorouracil (5-FU) showed no significant decrease in MPCE frequency. The data suggest that the induction of micronuclei by mutagens is inhibited by treatment with estrogen, and this could result in a sex difference in the sensitivity of mice employed in the micronucleus test. Mechanisms of the inhibitory effects of estrogen are discussed; these might include a suppression of erythropoiesis and a possible effect on the cell membrane permeability of erythroblasts.

A prospective, randomized, double-masked, and placebo-controlled study was performed to evaluate the effects of a localized and sustained release of mitomycin on the success of glaucoma filtration surgery in rabbits. A bioerodible polymer was used as the drug carrier. Full-thickness filtration surgeries were performed and data from 22 rabbits were collected. One eye received a polymer impregnated with mitomycin (0.02 mg or 0.06 mg), while the fellow eye received a drug-free polymer. Intraocular pressure, bleb survival, and postoperative complications were investigated. Intraocular pressures remained lower for a longer period of time (P less than 0.004) and filtration blebs lasted longer (P less than 0.05) in experimental eyes than in control eyes. Conjunctivitis and sectorial corneal haze occurred more frequently in eyes treated with the higher dosage mitomycin compared with control eyes. The use of mitomycin-C in a polymer delivery system appeared to promote the success of glaucoma filtration surgery in rabbits. With the lower dosage of mitomycin, clinically significant ocular toxicity was not noted.

Cytogenetic analysis was used to test whether or not human bile induced chromosome abnormalities in lymphocytes grown in culture. Bile was obtained from gallbladders resected for various reasons such as cholecystitis, cholelithiasis, polypus and cancers of the biliary tract, stomach and pancreas. After adding human bile to a final concentration of 25 microliters/ml or 12.5 microliters/ml, the culture medium was incubated at 37 degrees C for 72 hr. Air-dried slides were stained with conventional Giemsa and the numerical and structural chromosome abnormalities were scored. Positive and negative controls in terms of chromosome abnormalities were established by using 0.03 micrograms/ml mitomycin C (MMC) and 0.9% normal saline, respectively. Cytogenetic analysis was successfully performed in 6 out of 10 bile samples (60.0%). Bile alone did not induce numerical or structural chromosome abnormalities. Structural abnormalities increased significantly in the 25 microliters/ml bile + 0.03 micrograms/ml MMC group, compared with the 0.03 micrograms/ml MMC group: 36.0% vs. 20.7% in the chromatid-type gaps and breaks, 27.8% vs. 22.7% in the chromosome-type gaps and breaks, and 8.3% vs. 3.2% in the exchange-type abnormalities. It is likely that the interaction between bile and MMC is synergistic rather than additive.

We have previously reported the isolation of CHO cell lines resistant to mitomycin C under aerobic conditions of drug exposure. Here it is reported that these cell lines have the same response to mitomycin C under hypoxic conditions as do controls. The cells are shown to have lower levels of DT-diaphorase activity than controls, but similar levels of activity of NADPH:cytochrome c reductase, another enzyme involved in the metabolism of mitomycin C. Evidence for molecular defects in the DT-diaphorase gene or gene transcript is presented for the deficient cell lines. The consequences of this DT-diaphorase deficiency is further explored by testing the toxicity of menadione, an established enzyme substrate. The isolation of CHO cell lines deficient in DT-diaphorase activity and resistant to mitomycin C under aerobic but not hypoxic conditions suggests that mitomycin C reduction by this enzyme has a significant impact on cytotoxicity under aerobic but not hypoxic conditions. Similarly, DT-diaphorase metabolism of menadione does not appear to have a significant impact on cytotoxicity in CHO cells.

Coculture of two different cell lines in monolayer or spheroids was used to investigate the spontaneous transfer of dominant genes determining drug resistance. MGH-U1 human bladder cancer cells (ouabain-sensitive, mitomycin C-resistant) were cocultured with UV-20 cells (a subline of Chinese hamster ovary cells which is ouabain-resistant and mitomycin C-sensitive). We investigated the possible transfer of mitomycin-C resistance from human to rodent cells by selection in both ouabain and mitomycin C. Regardless of coculture conditions, the frequency of surviving cells was at a similar level to that expected from studies of cell survival when cells were cultured alone. We found no evidence of spontaneous transfer of drug resistance between the two cell lines.

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.

A wild type Chinese hamster cell line (AA8) and three repair-deficient sublines of AA8 (EM9, UV4, and UV5) were used to study the nature of the cytotoxic lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin under aerobic and hypoxic conditions. The sensitivities of the repair-deficient sublines to the drugs varied markedly: EM9 was similar to AA8, whereas UV4 was exquisitely sensitive and UV5 was of intermediate sensitivity. Moreover, both the relative toxicities of the two drugs and the relative toxicities of each drug under aerobic and hypoxic conditions varied for the different cell lines. These data suggest that there are differences in the spectra of toxic lesions produced by mitomycin and porfiromycin and that there are differences in the lesions produced by these drugs under aerobic and hypoxic conditions.

The radiosensitizing and cytotoxic properties of Mitomycin C (MMC) was investigated in vivo using regrowth delay and tumor control assays. MMC significantly enhanced the radiation-induced growth delay when administered 15 min before irradiation; the slope of the dose response curve significantly increased and corresponded to a Dose Modifying Factor (DMF) of 1.9 (1.5-2.3; p less than 0.001). When MMC was given 4 hr after irradiation, the additional regrowth delay resulted in a parallel shift of the dose response curve, and MMC was not significantly dose modifying (DMF 1.3 (0.9-1.3); p less than 0.05). From isobologram analysis it was found that the preirradiation MMC schedule resulted in supra-additive responses, whereas MMC given after irradiation had an additive effect. The enhancement of radiation-induced tumor control was similarly found to peak when MMC was given 6 hr to 15 min prior to irradiation. At these intervals, the observed TCD50 for the combined treatments relative to radiation alone corresponded to Enhancement Ratios of 1.27 and 1.29, respectively (p less than 0.001). Longer intervals between the modalities reduced the enhancement, but the combined treatments were still significantly better than radiation alone (ER 1.12, 1.16 and 1.17; p less than 0.001). The significant enhancement of tumor control correlated with a substantial drug-induced cytotoxic effect toward hypoxic tumor cells, as determined by clamped TCD50 experiments. A single dose of MMC (3 mg/kg) was found to kill up to 97% of all hypoxic tumor cells.

beta-Galactosidase activities (beta-GA) of E. coli strains carrying the fusion gene of recA and lacZ, GE94 and its DNA repair-deficient derivatives such as KY946[uvrA], KY945[recA] and KY943[lexA] treated with UV, 4NQO, MNNG and MMC were examined. The beta-GA, reflecting the SOS-inducing activity, of GE94 and KY946 treated with these compounds increased significantly with a clear dose-response relationship, and reached a maximum level within 60 min, while no response was seen in KY945 and KY943. Using KY946 and KY945 as a positive and a negative indicator, respectively, the SOS-inducing activity of oxidative mutagens, i.e., hydrogen peroxide (H2O2), formaldehyde, tert-butyl hydroperoxide, cumene hydroperoxide and streptonigrin, was investigated. Clear dose-dependent increases in beta-GA were observed in KY946 treated with all oxidative mutagens tested, but not in KY945. Significant increases in beta-GA were observed with a lower concentration of H2O2 and a shorter incubation time of 4NQO in this assay than in the umu-test. The assay, called 'Rec-lac test' by us, may be useful to detect environmental genotoxic substances.

Mitomycin C (MMC) is a bioreductive alkylating agent that is capable of generating oxygen radicals. Porfiromycin (PM) is an analog to MMC that generates oxygen radicals at a significantly lower level than the parent compound. Under aerobic conditions, the toxicity of MMC to EMT6 cells is 2.5-fold that of PM, whereas hypoxically the two are equitoxic. In the present studies, the protective effect of PZ-51 in combination with NAC was assessed against the dose-dependent toxicity of either MMC or PM under both aerobic and hypoxic conditions. Aerobically, the PZ-51 and NAC combination inhibited the toxicity of MMC at concentrations of between 0.25 and 2 microM but had no effect on PM toxicity. Under hypoxic conditions, the PZ-51 and NAC combination had no effect on either MMC or PM toxicity. These findings support a role for oxygen radical generation in the aerobic toxicity of MMC at clinically relevant doses.

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.

Adjuvant perioperative liver infusion chemotherapy with fluorouracil, mitomycin and heparin caused mild but significant myelosuppression in the first two postoperative weeks. This was associated with a slightly increased bleeding tendency. Mortality was not increased. Future trials might combine chemotherapy with hematopoietic growth factors in order to minimize myelosuppression.

To enable the treatment of hepatic metastasis with higher, theoretically more effective, doses of systemically toxic anticancer drugs, an isolated liver perfusion (ILP) technique was developed in WAG/Ola rats. First, in a toxicity study the maximally tolerated dose (MTD) of mitomycin C (MMC) was determined for a 25-min ILP and for hepatic artery infusion (HAI) after the administration of a bolus dose. The MTD in the ILP setting (4.8 mg/kg) was 4 times that using HAI (1.2 mg/kg). Subsequently, in a rat colorectal hepatic-metastasis model, concentrations of MMC in tumour, liver, plasma and perfusate were measured during a 25-min ILP to investigate the expected pharmacokinetic advantage of ILP. The mean plasma level determined after ILP (1.2 as well as 4.8 mg/kg MMC) was significantly lower (P less than 0.001) than that obtained following HAI. This may explain both the absence of severe systemic toxicity and the higher MTD in ILP-treated groups. No significant difference in mean tumour and liver tissue concentrations of MMC were found when the groups treated with 1.2 mg/kg drug via HAI vs ILP were compared. The mean MMC concentration in tumour tissue was significantly higher (almost 5 times; P less than 0.05) in rats treated by ILP with the MTD (4.8 mg/kg) than in those treated via HAI with the MTD (1.2 mg/kg). ILP of MMC can be safely performed using a dose 4 times higher than the MTD in the HAI setting, leading to an almost 5-fold concentration of MMC in hepatic metastasis. ILP of MMC may therefore represent a promising therapy for metastasis confined to the liver.

A computer controlled bench-top analyser was used to establish an automated in vitro-cytotoxicity test. It is based on the continuous monitoring of growth of Salmonella typhimurium TA 98 and TA 100 (strains that are also employed in the classical Ames test (1) for mutagenicity screening) under the influence of the toxic compounds. With this method we can quantify general cytotoxicity and in addition, some questions concerning the mechanism of the toxic influence on the test organisms can be answered. Its simplicity and reliability as well as the good correlation with in vivo data make the new test system suitable for pre-screening the toxicity of a large number of compounds within short time.

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.

Systemic toxicity is usually the dose-limiting factor in cancer chemotherapy. Regional chemotherapy is therefore an attractive strategy in the treatment of liver metastasis. Two ways of regional chemotherapy, hepatic artery infusion (HAI) and isolated liver perfusion (ILP), were compared investigating the difference in toxicity with tissue and biofluid concentrations of mitomycin C (MMC). In wistar derived WAG rats the maximally tolerated dose of mitomycin C via HAI was 1.2 mg kg-1. Body weight measurements after HAI with doses higher than 1.2 mg kg-1 suggest both an acute and delayed toxic effect of mitomycin C since the time weight curves were triphasic: a rapid weight loss, a steady state and a second fall in weight phase. These rats died due to systemic toxicity. ILP with 4.8 mg kg-1 was associated with no signs of systemic toxicity and only transient mild hepatotoxicity. ILP with 6.0 mg kg-1 was fatal mainly due to hepatic toxicity. The four times higher maximally tolerated dose in ILP resulted in a 4-5 times higher peak concentration of mitomycin C in liver tissue, while the plasma concentration remained significantly lower than in the HAI treated rats. In the tumour tissue a 500% higher concentration of mitomycin C was measured in the ILP with 4.8 mg kg-1 than in HAI with 1.2 mg kg-1 treated rats. We demonstrated that when mitomycin C was administered by ILP a 400% higher dose could be safely administered and resulted in a five times higher tumour tissue concentration. In view of the steep dose-response curve of this alkylating agent this opens new perspectives for the treatment of liver metastasis.

The combined effects of mitomycin C (MMC) and thio-tepa (TT) with gamma-ray doses of 5 and 9 Gy on mouse stem cells were studied using the spermatocyte test. Both chemicals induced very low yields of translocations after single treatments. In combined treatments with a dose of 5 Gy, a subadditive effect of MMC and an additive effect of TT were found. Combined with a dose of 9 Gy the compounds potentiated the effect of radiations. Up to now, most of the chemicals tested have shown additive effects when combined with doses of the ascending part of the dose-response curve and potentiating effects when combined with doses of its descending part. This has been considered additional confirmation of the concept that depletion of any kind of spermatogonia is sufficient to modify the genetic response of stem cells. However, the subadditive and additive responses found could be considered evidence that common biological mechanisms can modulate the response to combined treatments of chemicals and ionizing radiations.

A limited number of contradictory reports have appeared in the literature about the ability of radiofrequency (rf) radiation to induce chromosome aberrations in different biological systems. The technical documentation associated with such reports is often absent or deficient. In addition, no information is available as to whether any additional genotoxic hazard would result from a simultaneous exposure of mammalian cells to rf radiation and a chemical which (by itself) induces chromosome aberrations. In the work described, we have therefore tested two hypotheses. The first is that rf radiation by itself, at power densities and exposure conditions which are higher than is consistent with accepted safety guidelines, can induce chromosome aberrations in mammalian cells. The second is that, during a simultaneous exposure to a chemical known to be genotoxic, rf radiation can affect molecules, biochemical processes, or cellular organelles, and thus result in an increase or decrease in chromosome aberrations. Mitomycin C (MMC) and Adriamycin (ADR) were selected because they act by different mechanisms, and because they might put normal cells at risk during combined-modality rf radiation (hyperthermia)-chemotherapy treatment of cancer. The studies were performed with suitable 37 degrees C and equivalent convection heating-temperature controls in a manner designed to discriminate between any thermal and possible nonthermal action. Radiofrequency exposures were conducted for 2 h under conditions resulting in measurable heating (a maximum increase of 3.2 degrees C), with pulsed-wave rf radiation at a frequency of 2450 MHz and an average net forward power of 600 W, resulting in an SAR of 33.8 W/kg. Treatments with MMC or ADR were for a total of 2.5 h and encompassed the 2-h rf radiation exposure period. The CHO cells from each of the conditions were subsequently analyzed for chromosome aberrations. In cells exposed to rf radiation alone, and where a maximum temperature of approximately 40 degrees C was achieved in the tissue culture medium, no alteration in the frequency from 37 degrees C control levels was observed. Relative to the chemical treatment with MMC alone at 37 degrees C, for two different concentrations, no alteration was observed in the extent of chromosome aberrations induced by either simultaneous rf radiation exposure or convection heating to equivalent temperatures. At the ADR concentration that was used, most of the indices of chromosome aberrations which were scored indicated a similar result.(ABSTRACT TRUNCATED AT 400 WORDS)

Using 3-way differential staining (TWD) of sister chromatids, the fate of DNA lesions involved in sister-chromatid exchange (SCE) formation was determined in murine bone marrow cells in vivo, after treatment with either mitomycin C (MMC) or cyclophosphamide (CP). Both MMC (2.6 mg/kg b.w.) and CP (7 mg/kg b.w.) induced an SCE frequency near the expected in the 2 subsequent cell divisions, but the frequency of SCE occurring at the same locus in successive cell divisions was substantially lower than expected. The results are compared with previous data obtained after exposure to gamma-rays. A model of SCE induction is proposed.

The property of pluronic F127 (PLF) as a base for an anticancer agent, mitomycin C (MMC) was evaluated. A weighed amount of PLF and MMC were slowly added to cold water and mixed completely. The solution was warmed to 37 degrees C and MMC containing PLF gel was formed. The cumulative amount of MMC which was released from the gel during a 3-hour period was calculated. 5.5 percent of MMC was released from PLF-MMC gel which contained 20 or 25% PLF and 3.8% MMC from PLF-MMC gel containing 30% PLF. Intraperitoneal (i.p.) administration of large amount of PLF gel alone in rabbits or mouse revealed severe hepatorenal toxicity, and acute i.p. administered LD50 of PLF was supposed to be between 1.7 g and 5.0 g/kg body weight in mouse. Further evaluation of gradual releasing property of PLF is needed.

Studies were conducted to evaluate the effect of experimental protocol on the ability of benzidine (BZD), dimethylbenzanthracene (DMBA) and mitomycin C (MMC), administered by intraperitoneal injection, to induce micronuclei in polychromatic erythrocytes (PCE) of B6C3F1 mice. Three different treatment/sampling protocols were used, involving from one to three consecutive daily treatments and from three to one, respectively, consecutive daily samplings beginning 24 h after the last injection. DMBA and MMC elicited a significant micronucleus response in all three experimental protocols, while BZD induced a significant response only in the multiple injection protocols. Of the three protocols, the 3-day injection/single sample time protocol offers the greatest efficiency in minimizing the number of animals required in a study, in decreasing the time needed for scoring and in simplifying the statistical analysis. In addition, a comparison of the frequency of micronucleated PCE in peripheral blood and bone marrow following the treatment of mice with either BZD or DMBA suggests that, following a three injection protocol, either tissue can be used with equal efficacy.

Micronuclei (MN) can be formed by acentric chromosome fragments or whole lagging chromosomes. In order to discriminate MN produced by chromosome breakage from those arising from spindle malfunction a staining method using immunofluorescent kinetochore antibodies has been applied successfully in vitro. In the present study MN in polychromatic erythrocytes of mouse bone marrow cells induced in vivo by the spindle poison colchicine (COL) and the clastogen mitomycin C (MMC) were analysed after CREST staining. The staining method was modified by using pretreatment with detergents in order to allow a good penetration of the antibodies into the MN. About 66% of the MN induced by COL in contrast to only 4.5% of the MMC induced MN were CREST-positive. The preliminary results show that the CREST staining is also in MN induced in vivo capable of detecting the origin of MN and to discriminate between the spindle damaging or clastogenic activity of environmental agents. The method described will give supplementary information about MN, it cannot substitute for the common micronucleus test.

The frequencies of micronuclei (MNi) in the gill cells of the mussel Mytilus galloprovincialis were determined over a long period (up to 28 days) following a 48-h treatment with colchicine. The frequency of MNi at the end of treatment was significantly higher than in controls and 24 h later it had increased even more. After this period, the frequency of MNi rapidly declined until a plateau level was reached on day 2-3, which was significantly higher than the control baseline level, and persisted until the end of the experiment (28th day). In the same cell system we previously reported a persistence of an increased frequency of MNi after treatment with mitomycin C (MMC) (Majone et al., 1987). In order to establish the origin of MNi, the difference between their size distribution in MMC- and colchicine-treated animals was determined at the end of treatment as well as during the plateau phase. The difference was statistically significant (P less than 0.001) at the end of treatment, the MNi induced by MMC being smaller than those induced by colchicine. However, the difference during the plateau phase was not statistically significant. Human CREST antikinetochore fluorescent antibodies reacted with chromosome centromeres of Mytilus and were applied to gill cells at the end of a 48-h treatment with MMC or colchicine. About 60% of the MNi induced by colchicine but only 30% of those produced by MMC reacted positively with the fluorescent antibodies. This result indicates that the majority of MNi observed at the end of a 48-h treatment with MMC or colchicine originate, respectively, from acentric chromosome fragments and from whole lagging chromosomes.