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Helicobacter pylori (H. pylori) infection plays a role in the pathogenesis of peptic ulceration as well as active chronic gastritis. Possible mechanisms of H. pylori-induced mucosal injury include the generation of toxic monochloramine (NH2Cl) from oxidant (HOCl)--which is a product of activated neutrophils-and ammonia (NH3), which is a metabolite of H. pylori urease. To clarify mechanisms by which NH2Cl induces cytolysis, we determined the effects of glutathione (GSH) alteration and iron chelation on NH2Cl-induced damage in cultured rat gastric mucosal cells, because these are involved in oxidant injury. Cytotoxicity was quantified by chromium 51 release from prelabeled cells that were exposed to NH2Cl or hydrogen peroxide (H2O2). Although both NH2Cl and H2O2 caused a time-related and dose-dependent increase in 51Cr release, NH2Cl was more cytotoxic than H2O2. Pretreatment with extracellular GSH caused a right shift of the dose-response curve for NH2Cl, whereas pretreatment with diethyl maleate (a depletor of cellular GSH) rendered cells less resistant to NH2Cl. Iron chelation with 1,10-phenanthroline or deferoxamine failed to influence NH2Cl injury, whereas such treatment was protective against H2O2. Intracellular GSH seems to play an important role as a potent defense system against NH2Cl, as observed in H2O2-induced damage. However, the mechanisms of NH2Cl-induced damage seem to be distinctly different from cytolysis by H2O2 in terms of the mediation of cellular iron.

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.

The induction of gene mutations was examined in MutaMouse after an intraperitoneal injection of 7, 8-dimethylbenz[a]anthracene (DMBA) at 20 mg/kg in a collaborative study participated by four laboratories. Although the DMBA dose used was lower than the level that has been reported to induce micronucleated erythrocytes maximally in several mouse strains, a killing effect appeared after day 9 of the post-treatment interval. Mutations in lacZ transgene were detected by the positive selection assay following in vitro packaging of phage lambda from the genomic DNA of the transgenic animals that survived. The mutant induction was evaluated in the bone marrow, liver, skin, colon, kidney, thymus, and testis 7 to 28 days after the treatment. In the bone marrow, the mutant frequency reached a maximum, approximately a 30-fold increase, 14 days after the treatment and the increased frequency persisted at least up to day 28 of the post-treatment. Induction of mutants was detected in the liver, colon, thymus, and skin to lesser extents. Marginal responses were obtained in the kidney and testis. The slight increases in the mutant frequencies in the kidney and testis observed in some laboratories were within laboratory-to-laboratory or animal-to-animal variations. In contrast to the gene mutation induction in the bone marrow, the frequency of micronucleated reticulocytes increased transiently 3 days after the treatment and returned to a control level before day 8 of the post-treatment. It was suggested that DMBA induced gene mutation is fixed in stem cells depending on cell proliferation while DNA damages responsible for chromosome breakage are not transmitted to progeny cells.

The Non-genotoxic Carcinogen Study Group of the Environmental Mutagen Society of Japan organised the first step of an interlaboratory validation study on an improved cell transformation assay employing Balb/c 3T3 A31-1-1 cells. Nineteen laboratories participated in this study. The modified transformation assay was evaluated for its responsiveness, its interlaboratory reproducibility and its transferability. In this study, a mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12, supplemented with insulin-transferrin-ethanolamine-sodium selenite and 2% fetal bovine serum (FBS) was used during the period of expression of transformed foci, intead of the usual minimum essential medium with 10% FBS. 20-Methylcholanthrene (MCA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were selected as a prototype initiator and a tumour promoter, respectively. Two series of experiments were conducted. In the first series, the transformation activity of MCA was examined at various concentrations. In the absence of the promoting treatment with TPA, exposure to MCA only weakly induced transformed foci. In the presence of 0.1μg/ml TPA, all laboratories observed significant dose-dependent increases in the number of transformed foci with increasing MCA concentrations. In the second series of experiments, various concentrations of TPA were tested. In the absence of initiating treatment with MCA, exposure to TPA weakly induced transformed foci in about half of the laboratories. In the presence of 0.2μg/ml MCA, all the laboratories observed significant dose-dependent increases in the number of transformed foci with increasing TPA concentrations. The results from this study support the usefulness of this modified two-stage transformation assay with Balb/c 3T3 cells., (1999 FRAME.)

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by binding of its ligand, the proteolytic protein caspase-8 is recruited to a signalling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified by cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signalling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis.

Transgenic rodent mutation assays permit the detection and molecular analysis of various types of gene mutations, such as base changes and frameshifts, in a number of tissues. It is reported, however, that deletion mutations are not efficiently detected by the assays, in particular those using lambda phage shuttle vectors. Recently, a new transgenic mouse model, i.e., gpt delta, has been developed to selectively detect some types of deletions by Spi(-) selection. Spi(-) selection has an advantage over the other selections to preferentially identify deletions because only lambda phages deficient in both the red and gam gene functions are allowed to form phage plaques. In this study, we examined whether in vivo deletions induced by the treatment of mitomycin C (MMC) are detectable by the Spi(-) assay in the mouse model. The mice were treated with MMC (0.5, 1.0, 2.0, and 4.0 mg/kg, single intraperitoneal injection) and sacrificed 14 days after the dosing. The treatment at 4.0 mg/kg approximately doubled the mutant frequency of Spi(-) in the bone marrow, i.e., 2.52 x 10(-6) vs. 1.31 x 10(-6). The molecular analyses using polymerase chain reaction (PCR) and DNA sequencing indicated that seven Spi(-) mutants at 4.0 mg/kg group had deletions with molecular sizes from 0.8 kilo basepairs (kb) to 8.5 kb, whereas no such deletions were observed in the Spi(-) mutants in the control group. The results suggest that deletions induced by MMC in the bone marrow are efficiently detectable by Spi(-) selection and also that the molecular analyses are useful to evaluate the significance of a marginal increase in mutant frequency in the transgenic rodent mutation assays., (Copyright 1999 Wiley-Liss, Inc.)

To investigate how a carcinogenic promoter acts on cells mutated by an initiator, we used as a model, lacZ transgenic mouse and a positive selection system. Preliminary data for the mutational events in liver DNA of the mice was generated using diethylnitrosamine (DEN) and sodium phenobarbital (S-PB) as initiator and promoter, respectively. In our first experiment, male MutaMice received a single i.p. injection of saline or 100 mg/kg DEN and were fed a normal diet for 7 days and 500 p.p.m. S-PB in the diet for 21 days. Liver DNA was harvested after a 1 night fast on days 7 and 28 post-DEN treatment. In our second experiment, male mice received a single i.p. injection of phosphate buffered saline or 50 mg/kg DEN and were fed a normal diet for 7 days, a diet with S-PB for 14 days and then a normal diet for 7 days. Liver DNA was harvested after a 1 night fast on days 7, 21 and 28 post-DEN treatment. The S-PB diet enhanced absolute and relative liver weights in all groups. The single intraperitoneal dose of 50 or 100 mg/kg DEN induced high mutation frequencies (MF) in liver, lacZ genes on days 7, 21 and 28. There were no remarkable differences of the MF among any sampling days for animals receiving DEN and a normal diet. S-PB feeding at 500 p.p.m. for 21 days failed to affect the MF in groups given saline or 100 mg/kg DEN. On the other hand, when 50 mg/kg DEN was given, S-PB feeding at 500 p.p.m. for 14 days elevated the MF in liver DNA on days 21 and 28 to approximately 1.8 and 4.0 times the MF, respectively, of the mice fed the normal diet. Consequently, S-PB might preferentially promote certain initiated cells participating in a balance between cell death and proliferation.

The germ cell mutagens ethyl nitrosourea (ENU) and methyl methanesulfonate (MMS), were tested for their genotoxicity in sperm cells and testicular germ cells using lacZ transgenic mice (Muta Mouse). Eight- to 10-week-old Muta mice were treated with ENU (150 mg/kg) or MMS (40 mg/kg) by intraperitoneal injection. Three and 14 days after treatment, testes and sperm were collected for lacZ mutation analysis. Sperm were isolated from the epididymis and vas deferens by washing out the minced tissue. Germ cell DNA was isolated from testicular germ cells and sperm with the help of 2-mercaptoethanol, and the target lacZ gene, which is integrated into a lambda shuttle vector, was recovered by in vitro packaging. The resultant phages were allowed to infect to E. coli C (galE), and the lacZ mutant plaques were dominantly selected on a plate containing phenyl-beta-D-galactoside. Spontaneous mutant frequencies (MF) in vehicle-treated control mice were approximately 1 x 10(-5) and 3 x 10(-5) in testicular germ cells and sperm, respectively, at both sampling times. ENU treatment increased the MF in the testicular germ cells to 5 x 10(-5) on days 3 and 14, but did not affect sperm MF. MMS was not mutagenic in either tissue. The peripheral blood micronucleus assay was performed on the same animals 48 h after treatment, and strong inductions of micronucleated reticulocytes (MNRETs) were observed in both ENU- and MMS-treated mice. These data suggest that agents mutagenic to premeiotic germ cells, e.g., ENU, can be detected by transgenic mutation assay system using germ cells isolated from the testis. On the other hand, those mutagenic to postmeiotic cells, e.g., MMS, are insensitive in the assay system.

Unlabelled: Endoscopic variceal ligation (EVL) has been accepted as a new treatment for esophageal varices in cirrhotic patients, and evaluated to have a lower incidence of complications compared to sclerotherapy. Sclerotherapy may increase the risk of portal hypertensive gastropathy (PHG), which is recognized to be gastric mucosal congestion due to portal hypertension in cirrhotics. However, the gastric mucosal hemodynamics after EVL has not been established yet. The aim of this study is to assess whether EVL could affect the hemodynamics of the gastric mucosa., Patients and Methods: Eleven cirrhotic patients with severe esophageal varices, who underwent prophylactic EVL were enrolled in the trial. Age and sex-matched non-cirrhotic patients who had only gastric mucosal atrophy were entered as control to compare the mucosal hemodynamics to that of cirrhotic patient. EVL was performed as described by Stiegmann et al. The gastric mucosal hemodynamics was evaluated with both gastric mucosal blood volume (IHB) and hemoglobin O2 saturation (ISO2), which were measured by reflectance spectrophotometry during endoscopy. These parameters were measured in the three points of the stomach (gastric antrum, lower corpus and upper corpus) just before and after EVL., Result: In all patients with cirrhosis, mild PHG was observed endoscopically. ISO2 in cirrhotic patients was significantly lower in all points of the stomach compared with control. IHB in cirrhotic patients was not significantly different from control. ISO2 of post-EVL was significantly lower than that of pre-EVL, whereas IHB of post-EVL revealed significantly higher than that of pre-EVL. However, endoscopic grade of PHG remained mild. The hemodynamics of the mucosa two weeks after the initial EVL showed improvement of the congestion., Conclusions: The gastric mucosal hemodynamics showed increment of the gastric blood volume and decreased hemoglobin O2 saturation in cirrhotic patients, indicating that cirrhotic gastric mucosa is in congestive condition. EVL for esophageal varices makes the gastric mucosa more congestive soon after the procedure in spite of the same grade of endoscopic PHG. However, the worsened congestion improves within a few weeks.

Reactive oxygen metabolites may be involved in the pathogenesis of ethanol-, nonsteroidal antiinflammatory drug-, Helicobacter pylori-, or ischemia/reoxygenation-induced gastric mucosal injury in vivo. Iron plays a critical role in mediating oxidant injury in vitro. The present study examined a possible role of lipid peroxidation in inducing oxidant damage by determining the effect of iron chelation on cytotoxicity and on lipid peroxidation in cultured rat gastric cells. Cytotoxicity was quantified by 51Cr release from prelabeled cells that were exposed to tert-butyl hydroperoxide (tBHP) so as to overwhelm the glutathione redox cycle. Lipid peroxidation was assessed by measuring malondialdehyde (MDA) production. tBHP caused a time-related and dose-dependent increase of 51Cr release. The presence of phenanthroline (a chelator of Fe2+) during tBHP exposure and pretreatment with deferoxamine (a chelator of Fe3+) reduced tBHP-induced 51Cr release dose dependently. The generation of MDA increased as the concentrations of tBHP increased, but in a time course study, such generation preceded cytolysis. Both iron chelators attenuated MDA production in a dose-dependent fashion. Oxidant stress causes lipid peroxidation in cultured gastric cells, which is then followed by cytolysis. Iron plays a critical role in inducing lipid peroxidation as well as in mediating cytolysis. Iron chelation protects these cells from oxidant stress presumably through inhibition of lipid peroxidation.

Induction of micronucleated erythrocytes by a recombinant human erythropoietin (rhEPO) was examined using both in vitro and in vivo test systems. A small, significant and dose-related increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow of mice administered i.p. with 12,500-50,000 IU/kg rhEPO was induced at 48 h sampling time. A clear positive dose--response relationship and significant increase in the frequency of micronucleated reticulocytes (MNRET) in peripheral blood of mice administered i.p. with 400-50,000 IU/kg rhEPO was noted at 48, 72 and 96 h sampling times. Conversely, in bacterial reverse mutation tests, no noticeable increase of auxotrophic revertants was observed in Salmonella typhimurium, TA100, TA98, TA1535, TA1537, or Escherichia coli, WP2 uvrA-, by treatment with 188-6000 IU/plate of rhEPO, with or without S9 mix. Furthermore, rhEPO at 750-6000 IU/ml did not induce chromosomal aberrations in vitro in CHL cells or human peripheral blood lymphocytes in a direct method nor in a metabolic activation method. Moreover, chromosomal aberrations were not detected in bone marrow cells of CD-1 male mice, even at high rhEPO concentrations (100,000 IU/kg) in vivo. Consequently, it was concluded that errors in the process of enucleation or differentiation of the erythrocytes should be equally considered as possible mechanisms for the increased frequencies of MNPCE and MNRET alongside induction of DNA damage or errors in the process of DNA repair.

Gastric epithelium is exposed not only to oxidants generated within the lumen, but also to those produced by ischemia/reperfusion. This study examined the mechanism(s) of oxidant-induced injury to cultured rat gastric mucosal cells, and characterized the antioxidant profile of these cells. Hydrogen peroxide (H2O2), generated by glucose oxidase, damaged cells dose-dependently, as assessed by increased leakage of labeled 51Cr. Glucose oxidase-induced damage was prevented by exogenous catalase (but not by exogenous superoxide dismutase). Chelation of cellular iron with desferrioxamine or phenanthroline specifically protected cells against H2O2, whereas binding of extracellular iron with apotransferrin failed to. Disruption of the glutathione redox cycle at three independent sites rendered cells less resistant to H2O2, whereas inhibition of cellular catalase did not result in sensitization of cells to H2O2. In conclusion, (1) oxidant injury induced by extracellular H2O2 is mediated by intracellular iron; (2) extracellular superoxide is not involved in the damaging process; and (3) the glutathione redox cycle plays a principal role in detoxifying H2O2 as a cellular antioxidant in cultured gastric mucosal cells.

The nude (athymic) mouse was used as a novel test system for the evaluation of genotoxicity of genetic recombinant human erythropoietin (rhEPO). A fibroblast cell line derived from the kidneys of baby hamsters, BHK-21, or a subclone of BHK-21 transfected with an expression vector containing the human EPO gene, named BXE cells, were implanted in nude mice. The concentration of EPO in the plasma of mice bearing BXE increased in relation to the increase in the weight of the tumor formed from growth of BXE cells. Increased values of hematocrit (Ht), ratio of reticulocytes to erythrocytes (RET ratio) and the number of red blood cells in mice bearing BXE indicated that excessive hematopoiesis was occurring in the host. However, the concentrations of EPO in the plasma of the mice bearing BHK-21 did not increase in relation to the cell mass and consequently the Ht values and RET ratios in these mice were not affected. Marked increases in the frequencies of micronucleated polychromatic erythrocytes (MNPCE) and micronucleated RET (MNRET) were noted in the mice bearing BXE, although no chromosomal aberrations were found in the spleen and marrow cells of the same mice. The increased levels of RET, MNRET and MNPCE seemed to result from acceleration of erythroblastic maturation and proliferation by rhEPO. It is, therefore, concluded that errors in the processes of enucleation or differentiation of erythrocytes should be equally considered as possible mechanisms alongside errors in genetic repair processes for the increased frequencies of MNPCE and MNRET.

Comparative studies on the induction of micronucleated erythrocytes by three recombinant human erythropoietins (rhEPOs) such as epoetin alpha, epoetin beta, and SNB-5001, and by urinary human EPO (uhEPO) were carried out in vivo. Small but significant increases in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow of mice following a single intraperitoneal (i.p.) administration of each of the three rhEPOs and uhEPO (25,000 IU/kg) were observed at the 48 h sampling time. Clear, significant increases in the frequency of micronucleated reticulocytes (MNRETs) in the peripheral blood of mice similarly treated with the four hEPOs at 50,000 IU/kg were noted at the 48, 72 and 96 h sampling times. When mice were dosed with 50,000 IU/kg of each of the hEPOs once each day for 6 days and micronucleus preparations were made 24 h after the last administration, micronucleus induction in the bone marrow was not clearly demonstrated. As before, MNRETs in these mice were significantly induced in all cases. Consequently, it was concluded that induction of MNPCEs and MNRETs was a common characteristic of both native and genetically recombinant EPO.

The therapeutic doses of beta-stimulants in the treatment of bronchial asthma show wide variation among the drugs. In the present study, we tried to construct a system for prediction of the optimum clinical dose of beta-stimulants as oral dosage forms and aerosols using the data from preclinical pharmacological studies and pharmacokinetic data in humans. Values of EC50 (drug concentration that gives the half-maximum bronchodilator effect) in the in vitro studies based on isolated trachea of guinea pigs were collected from the literature. The ratios of the EC50 value of each drug to that of isoproterenol were used as the indicator of in vitro pharmacological activity. There were significant correlations between the EC50 ratios and the oral dose or the maximum plasma concentration after single oral administration, but the correlation coefficients were relatively small (r < .9). On the other hand, a linear log-log relationship was observed between the EC50 ratio and the maximum plasma unbound concentration (Cu(max)) (slope = 0.907, r = .955, p < 0.001). In the case of aerosols, a good correlation was observed between the EC50 ratio and the dose (slope = 0.770, r = .900, p < 0.01). The predicted concentration of beta-stimulants in the lung after aerosol administration tended to be higher than the Cu(max) after oral administration, suggesting the contribution of nonspecific binding in the lung tissue. These findings indicate the possibility of predicting the appropriate dose of beta-stimulants based on the preclinical pharmacological data and the pharmacokinetic data in humans.

DNA breaks and repair in mouse leukemia L1210 cells treated with 3 different types of cross-linkers, mitomycin C (MMC), 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) and SN-07 (a macromolecular antibiotic), were studied. Measured in D37 values, MMC gave the highest number of cross-links per lethal 'hit' directly after the 1-h treatment in the alkaline elution assay, followed by ACNU and SN-07. A good dose-response increase in induced interstrand DNA cross-linking frequency was observed in cells treated with 2.5-10 micrograms/ml MMC and with 10-100 micrograms/ml ACNU for 1 h with and without 24-h post-incubation. After 6-h post-incubation, the highest frequency of cross-linking was observed in cells treated with 2.5 micrograms/ml MMC and 30 micrograms/ml ACNU, while cross-link production continued in the cells treated with SN-07 for 12-h post-incubation. No significant increase in DNA breaks was observed in cells treated with MMC throughout 24-h post-incubation. The highest frequency of single-strand DNA breaks in cells treated with ACNU was observed immediately after the treatment and they disappeared after 6-h post-incubation. After 24-h post-incubation, a marked enhancement of the DNA breaks was observed in cells treated with SN-07 and the cells contained double-strand DNA breaks also. RNA synthesis was not affected in the cells treated with 10 micrograms/ml MMC and slightly inhibited to 70% of control in those treated with 100 micrograms/ml ACNU, while DNA synthesis in both cells was significantly inhibited after 24-h post-incubation. By contrast, both RNA and DNA synthesis were completely inhibited in cells treated with 8.0 micrograms/ml SN-07.

Reverse mutation (Ames) tests with Salmonella typhimurium TA98, TA100 and TA1537, and chromosomal aberration tests in vitro with a Chinese hamster fibroblast cell line (CHL), were carried out on fluorinated pyrimidine derivatives, such as 5-fluorouracil (5-FU), 1-(2-tetrahydrofuryl)-5-fluorouracil (FT), 5-fluorodeoxyuridine (FUdR), 1,3-bis(2-tetrahydrofuryl)-5-fluorouracil (FD-1) and a mixture of uracil and FT in the molar ratio 4 : 1 (UFT) (Fujii et al., 1978). For comparison, similar tests were also carried out on 4 anti-metabolic agents, a metabolite of FD-1 and a component of UFT, such as cytosine-1-beta-D-arabinofuranoside (AraC), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), 8-azaguanine (8-AG), 3-(2-tetrahydrofuryl)-5-fluorouracil (3-FT) and uracil. The anti-bacterial action of 4 fluorinated pyrimidine derivatives such as 5-FU, FT, FD-1 and UFT to TA100 was tested under the condition that buffer, S9 mix, S9 and albumin were present. 6-MP was only positive in the Ames test with TA100 in the system without S9 mix, while all others failed to show mutagenic activity. On the other hand, all compounds tested, except uracil, induced chromosomal aberrations on CHL cells in the system without metabolic activation. FT was degraded by S9, but there was no significant difference in the killing activity of FT among with buffer, S9 mix and albumin. The killing activity of 5-FU was the strongest with buffer, and it was slightly binding to albumin. The killing activity of 5-FU was mostly decreased by S9 mix. FD-1 showed the strongest anti-bacterial action when S9 mix was present but it was degraded by S9. UFT showed no anti-bacterial action in any conditions.

Antitumor activities of harringtonine(HA) and homoharringtonine(HO) both belong to cephalotaxus alkaloids were compared with those of vinca alkaloids, vincristine (VCR) and vinblastine (VLB). HA and HO had significant activities against P388 leukemia, L1210 leukemia and B16 melanoma by intraperitoneal injection comparable to VCR and VLB. The therapeutic index (LD10/ED50) of HA and HO in B16 melanoma system was 1.90 and 2.31, while those of VCR and VLB were 1.38 and less than 1.00, respectively. The oral administration of these cephalotaxus alkaloids evoked a significant antitumor activity but that of vinca alkaloids induced no activity against P388 and B16 melanoma. The efficacy of HO on various tumor systems was equal or greater than that of HA when comparison was made between these two cephalotaxus alkaloids. In contrast to VCR and VLB, HA and HO demonstrated moderate activity against P388/VCR, a subline of P388 resistant to vincristine. The intraperitoneal injection of VCR or VLB produced an increase in the mitotic index of P388 cells as compared with the control values, but HA or HO decreased it. HA and HO, cephalotaxus alkaloids are a new class of active compounds of plant origin which may differ from the vinca alkaloids in the mechanism of antitumor activity.

The modes of genotoxicity of a novel macromolecular antitumor antibiotic (SN-07) were examined using both prokaryotic and eukaryotic cells in vitro. The antibiotic induced a frameshift-type reverse mutation in Ames Salmonella typhimurium TA98 at 1.6-400 ng/plate with and without S9 mix. SN-07 also induced chromosomal aberrations and a forward mutation (6-TGr) in Chinese hamster V79 cells after 1 h treatment at 12.5-100 ng/ml without metabolic activation. The alkaline elution technique revealed that SN-07 induced interstrand DNA cross-linking dose-dependently after treatment with 2.5-10 micrograms/ml for 1 h followed by elution at pH 12.1, but it did not induce the dose-dependent cross-linking after the same treatment followed by elution at pH 12.6. It was also found that SN-07 induced single-strand DNA breaks (pH 12.1) and alkali-labile (pH 12.6) sites after treatment with 0.1-10 micrograms/ml for 1 h followed by 24-h post-incubation.

A novel macromolecular antibiotic SN-07 was obtained from the cultural supernatant of Actinomadura roseoviolacea var. miuraensis nov. var. The antibiotic was soluble in water, had a molecular weight of 18,000-22,000 daltons in 50 mM Tris-HCl buffer, pH 7.0, containing 2.0 M KCl as compared with authentic proteins. Its major constituents were nucleic acids. The substance had antibacterial activity against Gram-positive bacteria. It was also effective against lymphocytic leukemia P388 in vivo.

The mechanisms of action of a novel macromolecular antitumor antibiotic (SN-07) were examined using cultured mouse lymphoid leukemia L1210 cells. A shoulder exponential-type cytotoxicity was observed when the cells were treated with 3.13 to 100 ng/ml SN-07 for 1 hr and surviving colonies were counted after a 14-day incubation. It was found that 500 ng/ml SN-07 inhibited both RNA and DNA syntheses significantly at 40 and 80 min, respectively, while 8,000 ng/ml did not affect protein synthesis at 80 min. Treatment with a low concentration (80 ng/ml) of SN-07 for 1 hr inhibited both RNA and DNA syntheses after a 24-hr post-incubation. The alkaline elution technique revealed that 8,000 ng/ml SN-07 induced DNA interstrand cross-links time-dependently for 1 to 4 hr, and a 1-hr treatment with 80 to 8,000 ng/ml SN-07 induced DNA breaks after a 24-hr post-incubation. According to flow cytometric analysis, most L1210 cells progressed to the G2 phase in the cell cycle at a cytostatic concentration (25 ng/ml) of SN-07, and typical inhibition of the cell cycle progression was observed at a cytocidal concentration (200 ng/ml).