X-Y chromosome univalency in the testes of hyperthermic mice: I. Concomitant formation of multinucleated giant cells

Exposure of mice to an environment of approximately 35°C and 65% relative humidity for 2–5 days caused dissociation of the X-Y chromosome bivalent in diakinesis-metaphase I (primary) spermatocytes. Dissociation of autosomal bivalents, chromosome fragmentation, aneuploidy, and polyploidy were not significantly affected by heat stress. Heat stress also caused formation of multinucleated giant cells in the mouse testis. Giant cell formation followed a distribution similar to X-Y dissociation, reaching highest levels 5 days after heat stress and returning to control levels 50–70 days after heat stress. Spermatogonial activity was not permanently damaged by these conditions, and the germinal epithelium was able to repopulate. We believe that cell aberration or cell death resulting from either or both of these anomalies causes much of the decreased fertility found after subjecting scrotal mammals to high temperatures.     

X-Y chromosome univalency in the testes of hyperthermic mice: II. Light and electron microscopic analysis of synaptonemal complexes

Hyperthermia-induced X-Y dissociation has been observed in diakinesis-metaphase I sper-matocytes but not in pachytene spermatocytes, which have been implicated as highly susceptible to heat stress. To determine X-Y dissociation in pachytene spermatocytes and to compare levels of dissociation between pachytene and diakinesis-metaphase I spermatocytes male ICR mice were exposed to 35°C ± 0.07°C and 65% ± 0.14% relative humidity for 2 or 4 days. Control mice were housed at 24°C ± 0.04°C and 43% ± 0.58% relative humidity. Mice were killed 0, 3, 5, 8, or 10 days after stress and the testes processed for meiotic chromosome display at diakinesis-metaphase I and synaptonemal complex display at pachynema. Twenty-five to thirty cells per mouse at both stages of meiosis were observed with light microscopy, and pachytene spreads with transmission electron microscopy to determine heat-stress effects on synaptonemal complex structure. Statistical analyses revealed significant linear increases in X-Y dissociation with prolonged exposure to heat at pachynema and diakinesis-metaphase I. Levels of pachytene dissociation were one-half the levels of dissociation at diakinesis-metaphase I. The resolvable structure of the lateral elements of the synaptonemal complex was not affected by heat stress.     

Apoptosis and cell removal in the cryptorchid rat testis

In order to determine that apoptosis is responsible for large-scale germ cell elimination, we analyzed cells from cryptorchid testes both in histological sections and among those isolated in vitro. Apoptotic testicular cells during 3 to 7 days were only 8 to 30%, reaching a maximum of 80% by the end of 15 days of cryptorchidism. A similar trend was also observed with the number of dead cells. The process of large-scale germ cell removal in the initial stages was facilitated by the formation of multinucleated giant cells, which stained negative for apoptosis. Increase in oxidative stress and decrease in intratesticular testosterone was also observed. The above findings indicate that large-scale germ cell removal, at least during initial stages of cryptorchidism is not solely as a result of apoptosis. Declined intra testicular testosterone, elevated temperature and high oxidative stress following cryptorchidism probably affect cell viability and trigger a fast pace cell removal through giant cell formation.     

Polarized distribution of Na+,K+-ATPase in giant cells elicited in vivo and in vitro

Giant cell formation was analyzed to determine whether it results in the high level of Na+,K+-ATPase expression that characterizes multinucleated cells such as osteoclasts. Giant cells and fusing alveolar macrophages were subjected to morphological, immunological, and biochemical studies. Both subunits of the Na+,K+-ATPase were found to be present on the plasma membrane of giant cells. Their localization was restricted to the non-adherent domain of the cell surface. Dynamic studies of giant cell differentiation demonstrated that on culture and/or multinucleation, an increase in sodium pump alpha-subunit synthesis occurred and led to a high level of expression of Na pumps. Conversely, the adherent plasma membrane of giant cells was enriched in a lysosomal membrane antigen. This study demonstrates that culture and/or multinucleation induces a significant increase in the expression of sodium pumps. The polarized distribution of these pumps and of a lysosomal component suggests that fusing macrophages undergo biochemical and morphological alterations which prepare them for a new and specialized function in chronic inflammatory reactions. Giant cells may offer a suitable model system to study the differentiation of other related multinucleated cells, such as osteoclasts.     

X-Y chromosome dissociation in mouse strains difering in efficiency of spermatogenesis: Elevated frequency of univalents in pubertal males

Dissociation of the X-Y chromosome bivalent in diakinesis-metaphase I spermatocytes of adult mice was significantly more frequent in the CBA strain (29%) than in C57, KP, or KE strains (7–11%). Autosome dissociatio (1–5%) involved only the smallest chromosome pairs. Eleyatedfrequency of X-Y dissociation in the CBA strain correlates with significantly lower testes weight and lower yield of spermatogenesis, which suggests that sex bivalent dissociation man be responsible for some loss of spermatogenic cells. However, sperm quality is not affected, the percentage of normal spermatozoa and their fertlizing capacity being higher in CBA thatn in the remaining strains. Two congenic strains, KE and KE. CBA (the latter with the Y chromosome introduced from CBA), had the same level of X-Y dissociatios, suggesting that the Y chromosome plays no rle in the determination of this character. In comparison with adult males pubertal (27–29 day-old) males had twice as hig a frequency of X-Y dissociation in KE an KP strains, and combined frequeicies of dissociated sex and autosome bivalents were significantly higher in pubertal males of all tested strains. Although te level of chromosome dissociation is not sufficient to explain increased mortality of germ cells observed in pubertal males, it could be one of the contributing factors.     

Macrophages from human colostrum. Multinucleated giant cell formation by phytohemagglutinin and concanavalin A

Macrophages obtained from human colostrum were cultured in the presence of phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). PHA caused multinucleated giant cell formation which could be inhibited by the addition of N-acetyl-d-galactosamine. Con A caused multinucleated giant cell formation and was cytotoxic in higher concentrations. Both effects could be inhibited by addition of α-methyl-d-mannoside and α-methyl-d-glucoside. PWM did not cause multinucleated giant cell formation but was cytotoxic in high concentrations.     

Effects of thalidomide on reproductive function and early embryonic development in male and female New Zealand white rabbits

BACKGROUND : The present work was performed to determine the effect of thalidomide exposure on reproductive function and early embryonic development. METHODS : Twenty‐five female New Zealand White rabbits were orally gavaged with 0, 10, 50, or 100 mg/kg/day thalidomide 14 days prior to mating through to gestation day 7 for a total of 22 days. Treated females were Caesarean‐sectioned approximately 29 days after the date of attempted mating. Following mating with treated females, male rabbits (25/dose) were gavaged with 0, 30, 150, or 500 mg/kg/day beginning 14 days prior to mating with a group of untreated females (25/dose). Doses were administered through mating until the day before sacrifice for a minimum of 56 days. Untreated females were Caesarean‐sectioned 29 days after the last attempted mating. Comprehensive necropsy and histopathology of the reproductive system were performed. RESULTS : Treated females had reduction in body weight gain during gestation. Mating and pregnancy parameters were unaffected by thalidomide. At 100 m/kg, litter averages for corpora lutea, implantations, litter sizes, does with viable fetuses and live fetuses decreased and the number of early resorptions, does with any resorptions, does with all conceptuses resorbed, and the percent resorbed conceptuses per litter increased. The number of early resorptions, the average number of early resorptions per litter, and the percent resorbed conceptuses per litter increased at 10 and 50 mg/kg. There were no thalidomide‐related external fetal malformations. Mating and fertility in male rabbits were unaffected by thalidomide. There was an increased incidence of flaccid testes at 150 and 500 mg/kg and of bilateral small testes in all treated groups. At 500 mg/kg, there was degeneration of the germinal epithelium of the testicles with an increase in multinucleated giant cells in seminiferous tubule and a loss of round and elongating spermatids. CONCLUSIONS : Thalidomide had no adverse effects on mating and fertility in male and female rabbits dosed up to 500 and 100 mg/kg/day, respectively, for 14 days prior to mating. After 56 day of dosing, histopathologic changes with no associated sperm abnormalities were observed in the testicles. Embryonic development NOAEL for treated females mated to untreated males was <10 mg/kg. Corresponding fertility NOAEL for treated males mated to untreated females was 500 mg/kg. Birth Defects Res B 71:1–16, 2004. © 2004 Wiley‐Liss, Inc.     

Distinct Regions of the Large Extracellular Domain of Tetraspanin CD9 Are Involved in the Control of Human Multinucleated Giant Cell Formation

Multinucleated giant cells, formed by the fusion of monocytes/macrophages, are features of chronic granulomatous inflammation associated with infections or the persistent presence of foreign material. The tetraspanins CD9 and CD81 regulate multinucleated giant cell formation: soluble recombinant proteins corresponding to the large extracellular domain (EC2) of human but not mouse CD9 can inhibit multinucleated giant cell formation, whereas human CD81 EC2 can antagonise this effect. Tetraspanin EC2 are all likely to have a conserved three helix sub-domain and a much less well-conserved or hypervariable sub-domain formed by short helices and interconnecting loops stabilised by two or more disulfide bridges. Using CD9/CD81 EC2 chimeras and point mutants we have mapped the specific regions of the CD9 EC2 involved in multinucleated giant cell formation. These were primarily located in two helices, one in each sub-domain. The cysteine residues involved in the formation of the disulfide bridges in CD9 EC2 were all essential for inhibitory activity but a conserved glycine residue in the tetraspanin-defining ‘CCG’ motif was not. A tyrosine residue in one of the active regions that is not conserved between human and mouse CD9 EC2, predicted to be solvent-exposed, was found to be only peripherally involved in this activity. We have defined two spatially-distinct sites on the CD9 EC2 that are required for inhibitory activity. Agents that target these sites could have therapeutic applications in diseases in which multinucleated giant cells play a pathogenic role.     

Sodium o-iodobenzoate and hemoglobin-oxygen affinity: in vivo effects.

Sodium o-iodobenzoate (OISB) was given intravenously to 15 dogs to test the in vivo effect of this drug on the oxyhemoglobin dissociation curve. Administration of a single dose of 500 mg/kg was followed by an average increase in P50 (PO2 at 50% oxyhemoglobin saturation) of 3.6 mmHg from 26.8 +/- 0.5 to 30.4 +/- 1.8 mmHg (corrected to pH 7.4). This elevation was sustained for 7 days. During intravenous infusions of 200 mg/kg every other day for 3 wk, there was a sustained increase in P50 of 2.6 mmHg from 27.8 +/- 1.1 to 30.4 +/- 0.9 mmHg. All dogs survived the experiment and no ill effects of the drug were noted. An increase in serum lactate and pyruvate occurred in all animals following acute or chronic exposure to the drug. There was no significant change in whole blood pH, 2,3-diphosphoglycerate concentrations, intracellular pH, or serum total phosphate. Multiple infusions of sodium cyanate (50 mg/kg per day) reduced P50 by an average of 12.2 +/- 0.3 mmHg. A subsequent single infusion of OISB (500 mg/kg) failed to increase P50. Our preliminary data indicate that pharmacological manipulation of hemoglobin O2 affinity is possible with organic compounds unrelated to erythrocyte metabolism.     

Possible GABAergic Modulation in the Protective Effect of Zolpidem in Acute Hypoxic Stress-induced Behavior Alterations and Oxidative Damage

Hypoxia is an environmental stressor that is known to elicit alterations in both the autonomic nervous system and endocrine functions. The free radical or oxidative stress theory holds that oxidative reactions are mainly underlying neurodegenerative disorders. In fact among complex metabolic reactions occurring during hypoxia, many could be related to the formation of oxygen derived free radicals, causing a wide spectrum of cell damage. In present study, we investigated possible involvement of GABAergic mechanism in the protective effect of zolpidem against acute hypoxia-induced behavioral modification and biochemical alterations in mice. Mice were subjected to acute hypoxic stress for a period of 2 h. Acute hypoxic stress for 2 h caused significant impairment in locomotor activity, anxiety-like behavior, and antinocioceptive effect in mice. Biochemical analysis revealed a significant increased malondialdehyde, nitrite concentrations and depleted reduced glutathione and catalase levels. Pretreatment with zolpidem (5 and 10 mg/kg, i.p.) significantly improved locomotor activity, anti-anxiety effect, reduced tail flick latency and attenuated oxidative damage (reduced malondialdehyde, nitrite concentration, and restoration of reduced glutathione and catalase levels) as compared to stressed control (hypoxia) (P < 0.05). Besides, protective effect of zolpidem (5 mg/kg) was blocked significantly by picrotoxin (1.0 mg/kg) or flumazenil (2 mg/kg) and potentiated by muscimol (0.05 mg/kg) in hypoxic animals (P < 0.05). These effects were significant as compared to zolpidem (5 mg/kg) per se (P < 0.05). Present study suggest that the possible involvement of GABAergic modulation in the protective effect of zolpidem against hypoxic stress.     

Gap Junction Enhancer Increases Efficacy of Cisplatin to Attenuate Mammary Tumor Growth

Cisplatin treatment has an overall 19% response rate in animal models with malignant tumors. Increasing gap junction activity in tumor cells provides the targets to enhance antineoplastic therapies. Previously, a new class of substituted quinolines (PQs) acts as gap junction enhancer, ability to increase the gap junctional intercellular communication, in breast cancer cells. We examined the effect of combinational treatment of PQs and antineoplastic drugs in an animal model, showing an increase in efficacy of antineoplastic drugs via the enhancement of gap junctions. Mice were implanted with estradiol-17ß (1.7 mg/pellet) before the injection of 1×10⁷ T47D breast cancer cells subcutaneously into the inguinal region of mammary fat pad. Animals were treated intraperitoneally with DMSO (control), cisplatin (3.5 mg/kg), PQ (25 mg/kg), or a combining treatment of cisplatin and PQ. Cisplatin alone decreased mammary tumor growth by 85% while combinational treatment of cisplatin and PQ1 or PQ7 showed an additional reduction of 77% and 22% of tumor growth after 7 treatments at every 2 days, respectively. Histological results showed a significant increase of gap junction proteins, Cx43 and Cx26, in PQ-treated tissues compared to control or cisplatin. Furthermore, evidence of highly stained caspase 3 in tumors of combinational treatment (PQ and cisplatin) was seen compared to cisplatin alone. We have showed for the first time an increase in the efficacy of antineoplastic drugs through a combinational treatment with PQs, a specific class of gap junction enhancers.     

PM-induced cardiac oxidative stress and dysfunction are mediated by autonomic stimulation

Epidemiological studies show that increases in particulate air pollution (PM) are associated with increases in cardiopulmonary morbidity and mortality. However, the mechanism(s) underlying the cardiac effects of PM remain unknown. We used pharmacological strategies to determine whether oxidants are implicated in PM-dependent cardiac dysfunction and whether PM-induced increase in autonomic stimulation on the heart mediates cardiac oxidative stress and toxicity. Adult Sprague-Dawley rats were exposed to either intratracheal instillation of urban air particles (UAP 750 microg) or to inhalation of concentrated ambient particles (CAPs mass concentration 700+/-180 microg/m3) for 5 h. Oxidative stress and cardiac function were evaluated 30 min after UAP instillation or immediately after exposure to CAPs. Instillation of UAP led to significant increases in heart oxidants measured as organ chemiluminescence (UAP: 38+/-5 cps/cm2, sham: 10+/-1 cps/cm2) or thiobarbituric acid reactive substances (TBARS, UAP: 76+/-10, Sham 30+/-6 pmol/mg protein). Heart rate increased immediately after exposure (UAP: 390+/-20 bpm, sham: 350+/-10 bpm) and returned to basal levels over the next 30 min. Heart rate variability (SDNN) was unchanged immediately after exposure, but significantly increased during the recovery phase (UAP: 3.4+/-0.2, Sham: 2.4+/-0.3). To determine the role of ROS in the development of cardiac malfunction, rats were treated with 50 mg/kg N-acetylcysteine (NAC) 1 h prior to UAP instillation or CAPs inhalation. NAC prevented changes in heart rate and SDNN in UAP-exposed rats (340+/-8 and 2.9+/-0.3, respectively). To investigate the role of the autonomic nervous system in PM-induced oxidative stress, rats were given 5 mg/kg atenolol (beta-1 receptor antagonist), 0.30 mg/kg glycopyrrolate (muscarinic receptor antagonist) or saline immediately before exposure to CAPs aerosols. Both atenolol and glycopyrrolate effectively prevented CAPs-induced cardiac oxidative stress (CL(ATEN): 11+/-1 cps/cm2, CL(GLYCO): 10+/-1 cps/cm2, TBARS(ATEN): 40+/-6 pmol/mg protein, TBARS(GLYCO): 38+/-6 pmol/mg protein). These data indicate that PM exposure increases cardiac oxidants via autonomic signals and the resulting oxidative stress is associated with significant functional alterations in the heart.     

Effect of Physalis peruviana L. on Cadmium-Induced Testicular Toxicity in Rats

Cadmium (Cd) stimulates the production of reactive oxygen species and causes tissue damage. We investigated here the protective effect of Physalis peruviana L. (family Solanaceae) against cadmium-induced testes toxicity in rats. Twenty-eight Wistar albino rats were used. They were divided into four groups (n?=?7). Group 1 was used as control. Group 2 was intraperitoneally injected with 6.5 mg/kg body weight (bwt) of cadmium chloride for 5 days. Group 3 was orally treated with 200 mg/kg bwt of methanolic extract of physalis (MEPh). Group 4 was pretreated with MEPh before cadmium for 5 days. Changes in body and testes weights were determined. Oxidative stress markers, antioxidant enzymes, and testosterone level were measured. Histopathological changes of testes were examined, and the immunohistochemical staining for the proapoptotic (caspase-3) protein was performed. The injection of cadmium caused a significant decrease in body weight, while a significant increase in testes weight and testes weight index was observed. Pretreatment with MEPh was associated with significant reduction in the toxic effects of Cd as shown by reduced testicular levels of malondialdehyde, nitric oxide, and caspase-3 expression and increased glutathione content, and the activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and testosterone were also increased. Testicular histopathology showed that Cd produced an extensive germ cell apoptosis, and the pretreatment of MEPh in Cd-treated rats significantly reduced Cd-induced testicular damage. On the basis of the above results, it can be hypothesized that P. peruviana L. has a protective effect against cadmium-induced testicular oxidative stress and apoptosis in the rat.     

Optimum conditions for detecting hepatic micronuclei caused by numerical chromosome aberration inducers in mice

To ascertain an optimum condition for detecting micronuclei in the liver caused by numerical aberration inducers, either carbendazim (125-1000mg/kg, p.o.), colchicine (0.375-1.5mg/kg, i.v.), cytochalasin B (2.5-20mg/kg, i.v.), diazepam (3.13-25mg/kg, i.v.), noscapine (7.8-62.5mg/kg, i.v.), paclitaxel (1-100mg/kg, i.v.) or trichlorfon (18.75-150mg/kg, i.v.) was administered once to male Slc:ddY mice 1 day before or after partial hepatectomy (PH, Day 1). Five days after PH (on Day 6), hepatic micronuclei were determined in conjunction with classifications of the main nuclei and relative liver weights as a proliferative indicator or a dysfunction marker of cell division. Additionally, hepatocyte proliferation index (HPI) was calculated by using mono-, bi- and multinucleated cell counts. Treatment of mice with six compounds, except for colchicine, after PH showed higher incidence of micronucleated hepatocytes (MNH) than that before PH, and also increases in binucleated and multinucleated cells. Especially for carbendazim, diazepam, noscapine and trichlorfon, the dosing after PH was essential for the detecting numerical aberration. Colchicine evidently increased HPI and decreased relative liver weights without MNH induction on Day 6. On Day 8 when HPI and relative liver weights almost returned to the basal range, a significant increase in MNH was noted. This implied that the strong inhibition of colchicine on hepatocyte proliferation may obstruct the induction of MNH on Day 6. In conclusion, to detect the potential numerical aberration, exposure of mice to test chemicals should be performed 1 day after PH, during which enhanced proliferation of hepatocytes was seen, and it would be better to analyze the liver specimens on Day 6 or more post-PH.     

Cysteamine in combination with N-acetylcysteine prevents acetaminophen-induced hepatotoxicity.

N-Acetylcysteine (NAC) is protective against acetaminophen-induced hepatotoxicity primarily by providing precursor for the glutathione synthetase pathway, while cysteamine has been demonstrated to alter the cytochrome P-450 dependent formation of toxic acetaminophen metabolite. Mice administered acetaminophen (500 mg/kg) had elevations of serum alanine aminotransferase (ALT) to 273.0 +/- 37.5 and 555.8 +/- 193.4 U/mL at 12 and 24 h, respectively, after injection. Administration of cysteamine (100 mg/kg) or NAC (500 mg/kg) significantly reduced serum ALT activity (p less than 0.001). Reducing the dose of NAC or cysteamine by 50% greatly reduced their hepatoprotective effect while the co-administration of the reduced doses of NAC (250 mg/kg) and cysteamine (50 mg/kg) following acetaminophen overdose prevented elevation of serum ALT activity (39.2 +/- 1.17 and 32.5 +/- 5.63 U/mL at 12 and 24 h post-injection, p less than 0.001) and preserved normal mouse hepatic histology. Neither NAC (500 mg/kg), cysteamine (100 mg/kg), or the lower doses in combination of both agents were found to alter the half-life or peak levels of acetaminophen. Liver microsomal aryl hydrocarbon hydroxylase activity measured 24 h after drug administration was not significantly different between treatment groups and controls receiving only saline. These results indicate a possible role for the concomitant use of NAC and cysteamine in the prevention of hepatic necrosis following toxic doses of acetaminophen. Neither decrease in plasma acetaminophen levels nor depression of cytochrome P-450 enzyme activity appears to be the mechanism of protection when these doses of NAC, cysteamine, or both drugs together are administered with a toxic dose of acetaminophen in mice.     

Kinetics and characteristics of murine macrophage-lymphocyte interaction in presence of PHA: appearance of multinucleated giant cells in vitro

The appearance of cellular associations between macrophages and lymphocytes--which we have denominated macrophage-lymphocyte rosettes--and their kinetic formation in the presence of phytohemagglutinin (PHA) have been studied in B10 A (4R) mice. The greatest number of macrophage-lymphocyte rosettes was found from 6 to 12 hours after incubation with PHA. During this time, 42.38 +/- 10.70 of the total number of macrophages had lymphocytes attached to their membranes. This percentage decreased to 17.33 +/- 2.07% after 24 hours. The activation of macrophages after PHA treatment was tested by the phagocytic capacity of these cells. This activity increased significantly 24 hours after incubation. In our assay, an increase in the appearance of multinucleated giant cells when compared to controls was also observed. When the macrophages were lymphocyte depleted, the appearance of the multinucleated giant cells was significantly lower. The kinetics for these formations are also discussed.     

Involvement of ADAM9 in multinucleated giant cell formation of blood monocytes

Monocytes-macrophages are converted to multinucleated giant cells by stimulation with various cytokines, and osteoclasts are the multinucleated giant cells derived from a monocyte-macrophage lineage. However, at present, the fusion peptides have not been clearly identified in monocytes-macrophages. The ADAM are a family of transmembrane glycoproteins that have a role in various biological functions. Interestingly, fertilin-alpha, ADAM9, and ADAM11 have potential fusion peptides. In this study, which ADAM was specifically expressed in monocytes stimulated with anti-CD98 antibody or RANKL and which factor(s) was functioning in monocytes as a fusion protein were investigated. ADAM1, 8, 10, 12, 15, 17, 20, and 21 mRNAs are expressed in blood monocytes incubated with control antibody, anti-FRP-1/CD98 antibody, or RANKL + M-CSF, while ADAM2, 7, 11, 13, 19, 23, 29, and 30 mRNAs could not be detected in these blood monocytes. Expression of ADAM9 and ADAM10 mRNAs are enhanced by either RANKL + M-CSF or anti-CD98 antibody. The expression of ADAM9 and ADAM10 is also induced in blood monocytes by anti-CD98 mAb. An anti-ADAM9 antibody enhances CD98-mediated cell aggregation, while it blocks CD98-mediated and RANKL-mediated multinucleated giant cell formation. A hydroxamate-based metalloprotease inhibitor, SI-27, which is found to suppress ADAM9 activity, suppresses multinucleated giant cell formation. New protein synthesis is necessary for the expression of ADAM9 mRNA and genistein suppresses induction of ADAM9 mRNA. This is the first report that ADAM9 is involved in monocyte fusion, such as CD98-mediated and RANKL-mediated cell fusion of blood monocytes. Furthermore, AMAM9 is one candidate for a fusion peptide in blood monocytes.     

Effect of dose schedule of vitamin E and hydroxethylruticide on intestinal toxicity induced by adriamycin

CDF1 mice receiving Adriamycin, 12 mg/kg IP develop a toxic GI mucositis. The mean survival in CDF1 mice after Adriamycin injection was found to be 6.5 +/- 2.0 weeks and could be increased by alcohol or acetate Vitamin E pretreatment (with 2 g/kg qDx7d) to 22.06 +/- 12.3 weeks or by treatment with Venoruton after Adriamycin (qDx7 with 1.5 g/kg) to 23.7 +/- 12.7 weeks. Other schedules were ineffective or harmful. The ability of Venoruton to enhance survival when given after Adriamycin encouraged us to proceed to tumor bearing mice. The maximum survival with CDF1 mice bearing 5 X 10(6) L1210 cells was 1 +/- 0.2 week which could be increased to 2.17 +/- 0.8 weeks with optimal dose Adriamycin (10 mg/kg). Optimum survival with Venoruton and a single dose of Adriamycin was 2.45 +/- 0.91 weeks with Venoruton, 1.5 g, qd X 14, and 12 mg/kg Adriamycin. Treatment of L1210 bearing mice with Adriamycin, 10 mg/kg on days 1 and 8, yielded a survival of 2.23 +/- 0.7 weeks. An equitoxic regimen of Adriamycin, 11 mg/kg on days 1 and 9, plus Venoruton, 1.5 g, qd X 14, increased survival 30% to 3.08 +/- 2.9 weeks. Venoruton is a promising agent to increase the therapeutic index of Adriamycin.     

Multinucleation of macrophages with water-solubilized lignin derivatives in vitro

When macrophages derived from bone marrow were incubated with lignin derivative at 10 micrograms/ml, they formed multinucleated giant cells containing more than sixty nuclei. Cine-microscopic observation revealed that the formation of the giant cells was due to cell fusion. This phenomenon was specific for primary-cultured macrophages, and was not observed with either a macrophage cell line or established cell lines derived from other tissues.