Synthesis and identification of products derived from the metabolism of the carcinostatic 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by rat liver microsomes. Liver microsomal metabolism of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea in the presence of reduced nicotinamide adenine dinucleotide phosphate and O2 was shown to produce seven metabolites that included the parent urea. A cytochrome P-450-dependent monohydroxylation of the cyclohexyl ring occurred in 3 positions, cis-3, trans-3, and cis-4, and on the methyl group to form a trans-4-hydroxymethyl derivative. In addition, monohydroxylation of the 2-chloroethyl carbon attached to the N-1 urea nitrogen yielded an alpha-hydroxy metabolite. A ring-hydroxylated derivative remained unidentified while the structures of all other such derivatives were established by comparison with compound synthesized, purified by high-pressure liquid chromatography, and characterized by mass spectral and nuclear magnetic resonance analyses. It was tentatively concluded that some parent urea is formed by a cytochrome P-450 dependent reaction because of a requirement for reduced nicotinamide adenine dinucleotide phosphate and inhibition by CO. Microsomes from rats pretreated with phenobarbital showed about a 3-fold increase in hydroxylation rate while phenobarbital-treated mice microsomes were induced 8-fold. However, in both species, the induced hydroxylation rate was about 4 nmol/min/mg protein. When microsomes from phenobarbital-induced rats were used, a mixture of 80% CO:20% O2 decreased the rate of formation of all metabolites to 14% of that in 80% N2:20% O2.


Production of sister chromatid exchanges by various cancer chemotherapeutic agents. Various cancer chemotherapeutic agents have been examined for their ability to produce increases in sister chromatid exchanges. Those agents which had been shown previously to produce oncogenic transformation as well as chromosomal breaks also showed significant increases in sister chromatid exchanges. Those drugs which had not been shown to be oncogenic or clastogenic in cell culture produced no increases in sister chromatid exchanges. In general, concentrations which yielded increases in sister chromatid exchanges were considerably lower than those which had been shown previously to produce oncogenic transformation and chromosomal breakage. This was particularly true for the alkylating agents. Thus, we concur that examining increases in the production of sister chromatid exchanges may be an additional sensitive method for detecting potential mutagenic and/or oncogenic agents in our environment.


Phase 1 trial and assay of rubidazone (NSC 164011) in patients with advanced solid tumors. A new high-pressure liquid chromatographic method was developed for the simultaneous determination of rubidazone and daunorubicin in human plasma at concentrations as low as 60 ng/ml. Clinical toxicity and the stability of rubidazone were studied in nine patients with advanced solid tumors. Rubidazone was administered by i.v. infusion over 1 hr on a single day every 4 weeks. Moderate leukopenia was the dose-limiting toxicity in four of six patients treated at 150 mg sq/m. Assay of rubidazone in plasma samples obtained after administration of rubidazone showed that the drug was stable for at least 7 hr.


Volatile N-nitrosamines in the urine of normal donors and of bladder cancer patients. Volatile N-nitrosamines were detected in the urine of male volunteers with gas-liquid and high-pressure liquid chromatography interfaced to the thermal energy analyzer. Of 50 samples from normal males, 10 contained nitrosodimethylamine (0.02 to 0.10 micrograms/liter), 6 contained nitrosodiethylamine (0.02 to 3.10), 9 contained nitrosomorpholine (0.006 to 0.67), and none contained nitrosodibutylamine. Of 4 samples from bladder cancer patients, 2 contained nitrosodibutylamine (0.35 and 0.66). Cigarette smoking did not appear to be related to the pattern or amount of urinary volatile N-nitrosamines. The possibility that the N-nitrosamines arise from the diet or from endogenous production is considered.


Incidence and pathological features of spontaneous tumors in athymic nude mice. Systematic observation of 1141 nude mice (Swiss background [corrected] strain) that received human tumor transplants revealed 24 spontaneous tumors, 18 of lymphoreticular origin and 6 pulmonary adenomas. Spontaneous tumors were seen at an average age of 9.1 months, and 22 of the tumors were seen only in that fraction of our group (324 mice) surviving for 5 months or more (22 of 324 X 100 = 6.8%). Transplantation of these tumors to other nude mice was successful in three of five cases. Mice transplanted with adenocarcinoma of the colon and with tumors of the urogenital tract developed spontaneous tumors more often than did mice receiving other types of human tumor transplants. Progressive growth of the human tumor transplant occurred significantly less often in the mice that eventually developed spontaneous tumors than in the mice that showed no spontaneous tumor development. Nevertheless, the incidence of spontaneous tumors in these nude mice was similar to that reported for the thymus-bearing background strain.


Clinical status and rate of recovery of blood lymphocyte levels after radiotherapy for bladder cancer. Peripheral blood lymphocytes and leukocyte levels were monitored in 34 patients with bladder carcinoma before, during, and up to 5 years after radiotherapy. Radiotherapy in doses 6500 to 8500 rads caused a marked decline in the numbers of circulating leukocytes and particularly lymphocytes. In patients clinically free of disease for 5 years, lymphocyte counts returned to pretherapy levels within 3 years after radiotherapy. In contrast, in patients with recurrent or residual tumors lymphocyte counts failed to reach pretherapy levels within 3 years after therapy. The rate of recovery from radiation-induced lymphopenia was significantly different for patients who were free of disease as compared to those with recurrent or residual tumor (p less than 0.05). No correlation was found between posttherapy leukocyte levels and clinical status.


Glycosaminoglycans of normal and malignant cultured human mammary cells. Glycosaminoglycans have been characterized from a normal human breast cell line (HBL-100) and two different cell lines from human breast carcinoma (MDA-MB-231 and MCF-7). The glycosaminoglycans were labeled by exposure of cell cultures to [3H]glucosamine and [35S]sulfate and then isolated from both spent media and cells by pronase digestion and cetylpyridinium chloride fractionation. They were further characterized by (a) hexosamine composition, (b) controlled-pore glass exclusion chromatography, (c) reactivity with specific enzymes (hyaluronidase chondroitinase, heparitinase, and heparinase), (d) nitrous acid degradation, and (e) DEAD-Sephadex chromatography. The results indicate that the HBL-100 line synthesizes mainly hyaluronic acid, most of which is secreted into the medium. Chondroitin sulfate and heparan sulfate are the predominant glycosaminoglycans synthesized by the cancer lines; both are found mainly in the spent medium, but the hyaluronic acid synthesized by the MDA-MB-231 line remains cell associated. The cell-associated heparan sulfate had a molecular weight in excess of 13,000 and may contain linkages susceptible to testicular hyaluronidase. The MCF-7 cells produce significantly lower amounts of glycosaminoglycans than do the other two lines.


Activation of tumoricidal properties in mouse macrophages by lymphokines encapsulated in liposomes. Cell-free culture supernatants rich in macrophage-activating factor (MAF) activity obtained from mitogen-stimulated F344 rat lymphocytes have been encapsulated within liposomes of differing size and lipid composition and their ability to render normal mouse macrophages cytotoxic for tumor cells in vitro has been compared with that of unencapsulated (free) MAF added to the extracellular medium. Normal macrophages from C57BL/6, C3H/Hen, and C57BL/6 X C3H F1 mice treated with liposome-encapsulated MAF exhibited significant in vitro cytotoxicity against syngeneic and allogeneic tumor cells but did not kill nontumorigenic normal cells. Dose-response measurements revealed that liposome-encapsulated MAF was able to render macrophages tumoricidal at concentrations of at least 20,000 times lower than free MAF. Liposomes containing MAF were able to activate macrophages in the presence of p-nitrophenyl-2-O-alpha-L-fucopyranosyl-beta-D-galactopyranoside, a potent inhibitor of free MAF, indicating that encapsulated MAF was protected within liposomes and that liposome-mediated activation was not caused by small amounts of MAF released into the culture medium from "leaky" liposomes. Liposome-encapsulated MAF was also able to activate macrophages which were refractory to activation by free MAF following either removal of presumably surface receptors for MAF by pronase and/or alpha-L-fucosidase or occupation of the MAF receptor on macrophages by fucose-binding plant lectins (Ulex europaeus 1 and Lotus tetragonolobus agglutinins). Also, populations of nontumoricidal inflammatory tissue macrophages, which were inherently unresponsive to free MAF, would be rendered tumoricidal in vitro by incubation with liposome-encapsulated MAF. Collectively, the data suggest that MAF can render macrophages tumoricidal by acting on intracellular sites.


Mutually exclusive genetic signatures of human breast tumor cell lines with a common chromosomal marker. Seventeen recently established human breast carcinoma cell lines of metastatic origin (MDA series), HeLa cells, and MCF-7 (a well-established breast carcinoma cell line) were studied by starch gel electrophoresis for allozymic differences at 17 enzyme loci. Ten loci proved to be informative in establishing unique genetic signatures for all of the lines with the exception of MDA-134 and -309, which had the same genetic signatures. The probability of these latter two lines being of independent origin and finding their similar genetic signatures by chance is 0.07. These studies enable us to conclude that the chromosomal marker shown to be common to these breast carcinoma cell lines of metastatic origin is not present because of cross-contamination of the lines with other long-term lines or each other.


Increased susceptibility of mouse cells to fluorescent light-induced chromosome damage after long-term culture and malignant transformation. Exposure of mouse cells in culture to fluorescent light has been shown to produce chromatid breaks and exchanges. Hydrogen peroxide formed in the cell during illumination has been implicated as the causative agent. The present results indicate that susceptibility to light-induced chromosome damage increases with time in culture and seems to be associated with or requisite for the spontaneous malignant transformation of mouse cells. All three cell lines followed during long-term culture that either became tumorigenic or showed cytological evidence of neoplastic transformation developed a concomitant increase in susceptibility. In three additional cell lines, susceptibility to light-induced chromatid damage was significantly increased in the spontaneously transformed malignant cells as compared with their nonneoplastic precursors. The increased susceptibility is not simply the result of long-term culture, since three other nonneoplastic cell lines after prolonged culture were significantly less susceptible than their malignant counterparts. Increased susceptibility to light-induced chromatid damage could result from impaired DNA repair or from the loss of defense mechanisms for destroying H2O2 or scavenging free radicals.


Effects of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide, 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, and L-phenylalanine mustard on B16, Cloudman S91, and Harding-Passey mouse melanomas. The effects of 5-(3,3-Dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU), and L-phenylalanine mustard (L-PAM) have been compared by using three i.p. transplanted mouse melanomas: the B16 melanoma in C57BL/6 mice; the Harding-Passey (HP) malanoma in BALB/c X DBA/2F1 (hereafter called CD2F1) mice; and the Cloudman S91 melanoma in DBA/2 mice. HP melanoma responds well to all three drugs. S91 responds only to L-PAM and MeCCNU. DTIC may accelerate death in mice bearing this tumor. B16 responds well to L-PAM and moderately well to MeCCNU and to multiple injections of DTIC. The best response to DTIC and MeCCNU is given by HP, while the best response to L-PAM is given by S91. Tumor cell-doubling times were found to be 1.5 days for B16, 2 DAYS FOR HP, and 3 days for S91. HP would seem to be the most responsive malanmoma with respect to the 3 agents studied. This may be due to an interaction between the chemotherapeutic agents and the host immune response, since the HP tumor arose in a noninbred mouse and is thus nonsyngeneic with the CD2F1 host. All three tumors appear to be interesting biological models for studying drug combinations and combined therapeutic modalities against melanoma.


Fatty acid oxidation to H2O by Ehrlich ascites carcinoma in mice. Oxidation of free fatty acids (FFA) by Ehrlich ascites tumor in mice was studied in vivo by the direct measurement of 3H2O formed from [9,10-(3)]palmitate. The FFA tracer complexed to serum albumin was injected i.p. into unanesthetized mice, and blood plasma 3H2O was measured at different time points for 30 min. The contribution of 3H2O by desaturation of labeled palmitate to monounsaturated fatty acids in the tumor was estimated by the use of [1-14C]palmitate and was shown to be negligible during the course of our experiments. In order to estimate the rates of tumor FFA oxidation, the kinetics of the tumor-host water distribution system was studied by injecting tracer 3H2O i.p. and following the disappearance of 3H2O in the blood plasma at different time points for 30 min. The results of these experiments were used to compute the tumor FFA oxidation rate by multicompartmental analyses and SAAM. Despite the nearly anaerobic state of the ascites tumor fluid in vivo, cancer cells suspended in this fluid oxidized FFA at least as fast as they do in vitro under aerobic conditions. Moreover, according to our current estimate, the need of the tumor for FFA as a metabolic fuel appears to be much greater than its net lipid needs for growth.


Conditions for effective Bacillus Calmette-Guérin immunotherapy of postsurgical metastases of 13762A rat mammary adenocarcinoma. We evaluated critical variables in Bacillus Calmette-Guérin (BCG) immunotherapy of residual 13762A rat mammary adenocarcinoma. BCG was given intratumorally on Day 7 of tumor growth and followed by primary tumor excisions on Day 20. Untreated animals died on about Day 40 with axillary nodal and pulmonary parenchymal metastases. BCG-treated animals experienced prolonged survival, and some were cured. The highest dose (5.0 X 10(7) colony-forming units) of BCG was more effective than the lowest (0.5 X 10(7) colony-forming units), but 1,500 micrograms Corynebacterium parvum were more effective than even the highest BCG dose. Previous sensitization to BCG did not improve the effects of BCG treatment. BCG treatment was effective when given on Day 7 and sometimes as late as Day 12 or 17, but C. parvum was ineffective if given after Day 7. Repeated injections of BCG or C. parvum were not more effective than single injections were. Rats cured of residual 13762A tumor by BCG treatment were strongly and specifically immune to rechallenge. We concluded that a high dose (5.0 X 10(7) colony-forming units) of BCG given early (7 days) was the most effective presurgical treatment of 13762A metastases. Repeated injections or host presensitization to BCG did not improve the benefits.


Predictive value of cerebrospinal fluid polyamines in medulloblastoma. Seventy-five cerebrospinal fluid polyamine determinations were evaluated in 16 patients with medulloblastoma. Parameters utilized in evaluating patient status for correlation with the polyamine concentrations were neurological examination, computerized tomography, radionuclide scan, myelography, and cerebrospinal fluid cytology. Fifteen of the 16 patients showed absolute correlation with the eventual clinical picture. One determination on one patient resulted in a false negative. No false positives have been observed to date.


Inhibitory effect of prostaglandin F2 alpha on the growth of a hormone-dependent rat mammary tumor. Growth of the rat hormone-dependent mammary adenocarcinoma MTW-9 was inhibited by prostaglandin F2alpha (PGF2alpha) This inhibition of tumor growth was associated with a marked reduction in serum progesterone concentrations. The inhibitory effect of PGF2alpha on tumor growth was abolished by concomitant injections of progesterone. The growth rates of coimplanted prolactin-secreting pituitary tumor MtTW-5 and plasma concentrations of prolactin and estradiol were not affected significantly by PGF2alpha. Our data imply that the effect of PGF2alpha on growth of rat mammary adenocarcinoma is mediated by progesterone.


Distribution, frequency, and quantitative analysis of estrogen, progesterone, androgen, and glucocorticoid receptors in human breast cancer. The distribution and frequency of steroid hormone receptors are described 329 patients with breast cancer. The distribution of each of the steroid hormone receptors is unimodal with a progressive increase in the proportion of patients positive at lower receptor values. Receptor values expressed as fmol/mg cytoplasmic protein are well correlated with values expressed as fmol/mg breast tumor. Estrogen receptor was positive in 53% of the patients; progesterone receptor was positive in 38% of the patients; glucocorticoid receptor was positive in 52% of the patients; and androgen receptor was positive in 31% of the patients. The type of tissue assayed did not affect steroid hormone receptor positivity. For primary tumors, there was no correlation between steroid hormone receptor positivity and location of the tumor in the breast, size of the tumor, or extent of the disease. Each of the steroid hormone receptors was positively associated with each of the other steroid hormone receptors. Estrogen receptor was correlated with menopausal status and axillary nodal status, but these correlations did not exist for the other steroid hormone receptors. Estrogen receptor was not correlated with age after adjustment for menopausal status. The other steroid hormone receptors were not correlated with age.


Quantitative studies of radiation transformation with the A31-11 mouse BALB/3T3 cell line. A cloned mouse embryo-derived fibroblast cell line was used to study morphological transformation induced by X-rays and 254-nm ultraviolet light (UV). The transformation frequency increased exponentially with increasing dose from 10 to 400 rads for X-rays and 1.0 to 7.5 J/sq m for UV exposure. Split-dose X-ray exposures led to an enhancement in transformation at total doses below 100 rads and a reduction at doses of 300 to 400 rads. The induced transformation frequency varied among serum lots and was very dependent upon the initial cell density. Spontaneous transformants were observed in 10 of 22 consecutive experiments; the spontaneous transformation frequency was generally about 1 to 2 X 10(-5) as compared to induced frequencies which ranged up to 3 X 10(-3) for X-rays and 7.5 X 10(-4) for UV exposure. Further results indicate that this cell line has several potential advantages over the mouse 10T1/2 line for studies with relatively weak in vitro carcinogens such as radiation. These include (a) a reduced overall expression time for the appearance of transformed foci (4 weeks); (b) a high cloning efficiency (50 to 60%); and (c) the fact that about 20 times as many viable cells may be plated per dish for optimal results, allowing transformation frequencies as low as 10(-5) to be measured easily. On the other hand, there was more variability in the results among experiments with the 3T3 cell line.


Correlation of in vitro growth properties and tumorigenicity of Syrian hamster cell lines. Several in vitro phenotypic characteristics frequently associated with neoplastic cells were examined in a series of spontaneous and benzo(a)pyrene-induced Syrian hamster clonal cell lines which differed in their degree of tumorigenicity. Nonparametric statistical analysis demonstrated cloning efficiency in semisolid agar, enhanced fibrinolytic activity, decreased serum requirement for growth, decreased organization of intracellular actin, and increased cloning efficiency in liquid medium to be correlated with tumorigenicity. These correlations were not only qualitative but also quantitative. This suggests that the factors determining the degree of tumorigenicity of a cell can be cellular growth properties.


Endogenous tumor growth factor indicated by increased ornithine decarboxylase activity in malignant cells treated with host serum ascites fluid. Ornithine decarboxylase (ODC) production was used as an indicator of mitotic activity in neoplastic cells removed from murine hosts at progressive stages of growth. Cells from three ascites cancers and one fibrosarcoma were tested and showed declining ODC production with progressive growth. The cells were incubated with serum or malignant effusion fluid taken from the murine hosts at progressive stages of growth. For 2 to 3 weeks after tumor implantation, sera and, in particular, ascites fluids increasingly stimulated ODC production in cells at all stages of growth. With advancing disease, without the malignant growth having reached a stationary phase, the collected fluids decreasingly stimulated ODC production in the cells. The stimulating factor(s) in host serum and malignant effusion fluid were not tumor specific in the one combination tested.


Tumor regression after intralesional injection of mycobacterial components emulsified in 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene (squalene), 2,6,10,15,19,23-hexamethyltetracosane (squalane), peanut oil, or mineral oil. The influence of mineral oil, squalane, squalene, or peanut oil on the antitumor activity of emulsified Bacillus Calmette-Guérin cell walls or emulsified trehalose-6,6'-dimycolate was studied in mice, each with an established transplant of a syngeneic fibrosarcoma. Each animal received an intratumoral injection of Bacillus Calmette-Guérin cell walls (0.6 mg/mouse) or trehalose-6,6'-dimycolate (0.1 mg/mouse) emulsified in 1 to 10% oil. Emulsions of squalene or squalane but not peanut oil were effective substitutes for mineral oil as carriers of Bacillus Calmette-Guérin cell walls in the treatment of the tumor. Trehalose-6,6'-dimycolate was therapeutically active when it was incorporated in any of these four oils. The number of animals in which tumor regressed completely depended on the concentration of oil in the emulsion.


Prognostic factors in metastatic breast cancer treated with combination chemotherapy. Six hundred nineteen patients with metastatic breast cancer, treated with a combination of 5-fluorouracil, Adriamycin, and cyclophosphamide, or close variations of this program, with or without immunotherapy were analyzed retrospectively to identify those host, tumor, or treatment characteristics that might be of prognostic importance in predicting response to chemotherapy and survival from onset of the 5-fluorouracil-Adriamycin-cyclophosphamide treatments. Primary tumor characteristics such as size of primary, number of axillary nodes involved, stage at diagnosis, and type of surgery used for primary treatment were not found to be of prognostic significance. Host characteristics such as age, menstrual status, or family history of breast cancer were similarly unrelated to outcome. Non-Caucasian patients had a lower response rate and somewhat shorter survival than did Caucasians. Pretreatment weight loss, poor performance status, and abnormal biochemical and hematological values were of adverse prognostic significance. An estimate of total extent of disease based on criteria for rating extent of involvement at 12 potential sites was a much more important prognostic factor related to response and survival than actual sites of involvement or the traditional "dominant site" classification. There was a trend, however, for patients with bone involvement to have a longer survival than did patients with metastases to other organ sites. Shorter survival times were observed among patients exposed to extensive prior radiotherapy and those who failed to respond to prior hormonal treatment. The prognostic variables identified in this paper should be used for the design and comparison of clinical trials in the future.


Activity of a novel anthracenedione, 1,4-dihydroxy-5,8-bis(((2-[(2-hydroxyethyl)amino]ethyl)amino])-9,10-anthracenedione dihydrochloride, against experimental tumors in mice. 1,4-Dihydroxy-5,8-bis(((2[(2-hydroxyethyl)amino]ethyl)amino))-9,-10-anthracenedione dihydrochloride (CL 232315; NSC 301739D), a representative of a new chemical class of compounds with antineoplastic properties, has been evaluated for antitumor activity in experimental mouse tumor systems. The compound produced significant increases in life span (ILS) and long-term survivors when tested against the P388 and L1210 leukemias as well as the solid neoplasms, B16 melanoma and Colon Tumor 26. The optimal treatment regimens resulted in a 173 to greater than 200% ILS with 20 to 80% 60-day survivors in mice with P388 leukemia, A 205% ILS with 55% 60-day survivors in mice with L1210 leukemia, and an ILS of greater than 300% with 80% 90-day survivors in mice with B16 melanoma. In contrast to Adriamycin, CL 232315 was active against the i.v. implanted L1210 leukemia and demonstrated moderate activity against P388/Adria, a subline of P388 resistant to Adriamycin. The compound was ineffective when tested against the Lewis lung carcinoma and the Ridgway osteogenic sarcoma. CL 232315 was active i.p., s.c., and i.v., but p.o. activity was not demonstrated. Schedule dependency was not observed when the compound was administered once daily for nine days, once every four days, or as a single dose.


Effect of microtubule-associated proteins on the interaction of vincristine with microtubules and tubulin. The influence of microtubule-associated proteins on the interaction of vincristine with microtubules and tubulin was examined. Microtubules containing associated proteins were prepared in vitro from tubulin preparations which contained the proteins or from pure 6S tubulin and isolated microtubule-associated proteins. The presence of the associated proteins caused microtubules to be converted to stable spiral structures upon reacting with vincristine. When the proteins were absent, spirals were not formed, and the microtubules were completely disassembled by vincristine. At 0 degrees, 6S tubulin was converted to amorphous aggregates by vincristine, whereas if the associated proteins were present spirals were formed.


Soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA of Novikoff hepatoma cells. The nature of soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA was studied in Novikoff hepatoma cells. The decreased activity in hepatoma preparations was due to loss of a high-molecular-weight heat-labile factor. Although this factor cochromatographed with arginase activity on Sephadex G-150, it does not appear to result from this activity as judged by the failure of arginine to prevent the inhibitory effect on [3H]thymidine incorporation. Both liver and hepatomas contained a heat-stable factor with inhibitory activity. Studies with ethanol-soluble material suggested that the action was not solely attributable to the presence of unlabeled thymidine, since the apparent molecular weight was too high and since the factor(s) inhibited [3H]leucine incorporation into protein in addition to inhibiting [3H]thymidine incorporation in DNA.


Isolation and immunochemical characterization of antibodies from the sera of cancer patients which are reactive against human melanoma cell membranes by affinity chromatography. Immunoglobulins were isolated by affinity chromatography from sera of two patients with melanoma, one with sarcoma, and one with carcinoma. The affinity columns were prepared by covalently linking the membrane-rich fraction of biopsied melanoma cells to cyanogen bromide-activated agarose beads. The membrane-rich fractions were prepared by two methods: (a) hypotonic cell lysis, and (b) homogenization and differential centrifugation. Melanoma sera were autologous to melanoma membrane preparations. The isolated immunoglobulins showed immunoreactivity against antigens prepared from melanoma, sarcoma, and carcinoma cells by complement fixation but not against antigens prepared from normal human liver and lung tissues. Absorption of the isolated immunoglobulins with rabbit anti-human immunoglobulin immunobeads resulted in complete elimination of the complement-fixing antibody titer in one instance, whereas reduction occurred in other samples. Similar absorption with rabbit anti-human immunoglobulin M immunobeads resulted in reduction, but not complete elimination, of the antibody titers against target tumor cell preparations. These results suggest the presence of immunoreactive immunoglobulin G in all immunoglobulins and immunoglobulin M in some. Absorption of the isolated immunoglobulins with cultured sarcoma cells reduced but did not completely abolish antibody activity against autologous or allogeneic melanoma target antigen, whereas it did completely abolish activity against sarcoma target antigen. However, absorption with cultured allogeneic melanoma cells abolished the antibody activity against melanoma as well as sarcoma target antigens. The antibody titers of the isolated immunoglobulins were not affected by absorption with cultured lymphoblastoid cells. Since cultured melanoma and sarcoma cells were known to contain oncofetal antigen(s), these results suggest that the isolated immunoglobulins from cancer sera by melanoma membrane affinity chromatography were of at least two specificities: (a) antioncofetal; and (b) antitumor associated. The former group may be comprised of antibody to cross-reactive antigens associated with different histological types of tumors. However, it was apparent that a portion of the antibody activity was against common tumor-associated antigen(s). These results provide further evidence for the presence of common antigen(s) associated with biopsy specimens of human malignant melanoma.


Excision repair of cis-diamminedichloroplatinum(II)-induced damage to DNA of Chinese hamster cells. Platinum was lost from the DNA of cis-diamminedichloroplatinum(II) [cis-Pt(II)]-treated exponentially growing Chinese hamster V79-379A cells with a half-life of 28 hr. By contrast, platinum was lost from the DNA of cells treated in stationary-phase culture with a half-life of 4 days. Cells treated in and allowed to remain as a stationary-phase culture maintained an intact and apparently viable appearance. When the stationary-phase culture was diluted with fresh medium, cell division occurred, and cell survival, as measured by colony-forming ability, could be determined. Dilution of cells immediately after treatment with 40 micron cis-Pt(II) resulted in 0.19% control survival. There was an increase in the ability of the stationary-phase cells to survive cis-Pt(II) damage with time after treatment. Thus, after 3 days, the survival had increased from 0.19 to 15.9%. We demonstrate that this increased ability of stationary-phase cells to survive with time after treatment is due to DNA excision repair and hence that survival is inversely related to the extent of reaction of cis-Pt(II) with the DNA.


Increased excision of O6-methylguanine from rat liver DNA after chronic administration of dimethylnitrosamine. Male BDIV rats were given dimethylnitrosamine (2 mg/kg daily) by stomach tube on weekdays for a total of 9 weeks, the final dose being of 14C-labeled material. Control rats received only the labeled dimethylnitrosamine. Liver DNA was isolated at various times later (from 2 to 12 hr), and normal and alkylated purines were determined after hydrolysis in mild acid by chromatography on Sephadex G-10. The levels (measured as dpm/micronmol of parent base) of 7-methylguanine in the DNA of the pretreated rats were the same as or slightly higher than those of the control animals, and the persistence of this product was similar in both groups. This was also true for 3-methyladenine. In contrast, the initial amount of O6-methylguanine in the liver DNA of the pretreated rats was one-third of the amount found in the control rats, and the rate of loss of this product from DNA was higher in the pretreated animals. These differences were reflected in the alkylation product ratios: the 3-methyladenine:7-methylguanine ratios were closely similar in the two groups of animals at all times, whereas the O6-methylguanine:7-methylguanine ratio was initially 3 times higher in the control animals and fell more slowly. DNA synthesis (as measured by the incorporation of [3H]thymidine) was higher in the liver, kidney, and lung of rats receiving dimethylnitrosamine pretreatment. These findings are discussed with respect to the hepatocarcinogenicity of chronically administered dimethylnitrosamine.


Growth and treatment of Ehrlich tumor in mice with alloxan-induced diabetes. Hypoglycemia and hypoinsulinemia accompanied i.p. or i.m. growth of the Ehrlich tumor in CBA/H and BALB/c mice. Simultaneously, insulin accumulated in the ascitic fluid of tumor-bearing mice. In hosts rendered diabetic by means of alloxan, the tumor decreased the blood glucose almost to the level seen in nondiabetic mice. Tumor growth was retarded in diabetic hosts, but cells from such tumors, transplanted into secondary diabetic recipients, grew faster than in their primary diabetic hosts, similarly to "nondiabetic" tumor cells growing in nondiabetic hosts. This phenomenon of "adaptation" of the tumor to the diabetic state was prevented if diabetic tumor-bearing mice were daily treated with insulin. The tumor did not grow in all diabetic recipients; the frequency of takes correlated with severity of the diabetes, i.e., with the dose of alloxan given to induce it. The greater the dose, the less mice accepted the tumor. Insulin injection into diabetic tumor-bearing mice promoted the tumor growth. Simultaneous treatment of diabetes and the tumor afforded the best antitumor effect.


Identification of benzo(a)pyrene metabolites by gas chromatograph-mass spectrometer. A gas chromatograph-mass spectrometer was used in an attempt to achieve rapid separation and accurate identification of benzo(a)pyrene and its synthesized derivatives. All derivatives, after being trimethylsilylated, were developed on Dexsil-300 and OV-1 columns. The seven diols and four stereoisomeric 7,8,9,10-tetraols studied were separated successfully. The separation of the 12 phenols was unsatisfactory; 8- and 11-isomers appeared separately but the other 10 isomers made 3 peaks on the OV-1 column. Among the five derivatives reported to be present in animal tissues, 6-, 7-, and 9-phenols were separated, but 1- and 3-phenols were fused on the Dexsil-300 column. Quinones were converted to related dihydroxyl derivatives under silylation. The chromatographic separation of four of the six dihydroxyl derivatives was successful on the OV-1 column, but the 6,12- and 7,10-isomers remained in a single peak. The two diol-epoxides were unstable under silylation and therefore were detected by their breakdown products, 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene and tetraols. Data are listed on the mass spectra and retention times of all benzo(a)pyrene derivatives studied.


Alteration of hexosaminidase isozymes in human renal carcinoma. The activity and isozyme patterns of hexosaminidase in human renal carcinoma were studied in comparison with those of normal kidney. Hexosaminidase in extracts from normal kidney and renal carcinoma tissue could be separated into two major forms [hexosaminidase A (Hex A) and hexosaminidase B (Hex B)] by Cellogel electrophoresis or by diethylaminoethyl cellulose column chromatography. All of 10 renal carcinoma tissues showed a low activity ratio of Hex A to Hex B, as compared with the ratio in normal kidney; the ratio in renal carcinoma tissue was between 0.61 and 2.21 (mean, 1.30), while that in normal kidney was between 2.50 and 4.52 (mean, 3.46). Hexosaminidase activity and the ratio of Hex A to Hex B in renal carcinoma tissue were independent of the cell type and the differentiation grade of carcinoma tissue. Hex A and Hex B of renal carcinoma tissue differed from each other in physicochemical properties such as pH dependence of enzyme activity, thermostability, and Km's for two synthetic substrates, but each isozyme maintained its same physicochemical properties whether from normal or from carcinoma tissue. The isozyme patterns of cultured renal carcinoma cells and placenta were similar to those of the carcinoma tissue. The results presented here indicate that hexosaminidase isozymes in renal carcinoma tissue express at least oncoplacental patterns.


Estrogen and progesterone receptors in human breast cancer with concomitant assay of plasma 17beta-estradiol, progesterone, and prolactin levels. The specific estrogen receptor in the cytosol of human breast cancer tissue was assayed in 217 primary cases. The specific progesterone receptor was also assayed in 48 cases as evidence of estrogen action on the tissue. Both receptors were positive in 45.8% of all cases. Plasma 17beta-estradiol and progesterone were assayed concomitantly with these steroid receptors. The higher hormone levels were found in the cases with fewer receptor binding sites. The relationship between 17beta-estradiol levels in tumor cytosol and the number of binding sites was more clearly observed. Plasma prolactin levels, however, showed no correlation with the number of receptor binding sites or the plasma levels of sex steroids. None of these assayed substances had a clear correlation with the histological type of tumor or the clinical stage of the disease.


Killing of tumor cells in vitro by macrophages from mice treated with synthetic dehydrodipeptides. Treatment of NMRI mice i.p. with dehydrodipeptides [acetyldehydro-3-(2-thienyl)alanyltyrosine (SI); acetyldehydro-3-(2-furyl)alanyltyrosine (SII)] rendered macrophages cytolytic for several tumor cells in vitro. Normal peritoneal mouse macrophages from untreated mice not given injections of the peptides or from control mice given injections of phosphate-buffered saline were not cytotoxic. Moreover, supernatants from these in vivo-activated mouse peritoneal macrophages significantly increased the release of the cytoplasmic enzyme lactate dehydrogenase from freshly added target cells, showing that these cells had been killed. The macrophage activation to lyse tumor cells was sharply dose dependent and appeared about 48 hr after injection of the peptides. Although dehydrodipeptide SI was active in vivo at concentrations as low as 500 microgram/mouse, the same substance lacked activity in vitro at all concentrations tested up to 800 microgram/ml. Dehydrodipeptides activate macrophages through a T-cell-independent process to lyse tumor target cells. Macrophages from athymic nude (nu/nu) mice were less cytotoxic, but they still were stimulated; and the culture supernatants could kill about 50% of the tumor cells used. There are indications for a relative specific structure-activity relationship of dehydrodipeptides for inducing cytotoxic macrophages.


Effects of CCNU on hematopoiesis in rats. The effect of orally administered CCNU on proliferating and cytokinetically "resting" bone marrow cells was studied in rats. The resting bone marrow cells were selectively labeled with 3H-thymidine by the "complete 3H-thymidine labeling method." The results show a reduction in the number of absolute bone marrow cells to about 40% of the original value in 3 days. Regeneration was completed only after 22 days. No difference was found in the destructive or the regenerative pattern of CFU-C, myelopoietic, erythropoietic, lymphatic, or megakaryocytic cells. Resting and proliferating cells of the bone marrow were equally affected by CCNU.


Hypersensitivity reactions to iv melphalan during treatment of multiple myeloma: Cancer and Leukemia Group B experience. Ten patients developed allergic reactions to iv melphalan (L-PAM) during therapy for multiple myeloma. The incidence of such reactions was 2.4% among 425 patients receiving iv L-PAM with or without other drugs and 3.9% among 255 patients receiving iv L-PAM alone. Only one such reaction was demonstrated in 294 patients who initially received oral L-PAM. The median day of first reaction to iv L-PAM was Day 222 (range, Days 44-909) and the median total dose prior to a reaction was 185 mg (range, 51-250 mg). Of five patients who subsequently received oral L-PAM, four developed a reaction similar to that experienced with the iv drug.


Chemotherapy for sarcoma of the stomach. The results of chemotherapy for patients with advanced leiomyosarcoma of gastric origin were analyzed. A total of 23 single-agent and multi-drug regimens were given to 17 patients. DTIC and adriamycin (ADR) were the most commonly used chemotherapeutic agents. ADR was used alone or in combination in 13 patients, resulting in partial responses in two patients and disease stabilization in seven. One of the partial responses was obtained with the ADR-DTIC combination and the other was obtained with the cyclophosphamide-vincristine-ADR-DTIC combination. The survival duration for the responders was significantly longer than that obtained for those patients who had disease stabilization or progression. The results of the study show that leiomyosarcoma of gastric origin is less sensitive to chemotherapeutic agents than leiomyosarcomas of other sites, as reported in the literature. New approaches based on the natural history of the disease and with potential to improve the results of treatment of gastrointestinal sarcomas are indicated.


Experimental antitumor activity of aminoanthraquinones. The activity of a number of substituted alkylaminoanthraquinones was compared in transplanted murine tumor systems including P388 and L1210 leukemias, B16 melanoma, and colon carcinoma 26. The structure-activity relationships among this class of compounds are discussed. Several derivatives had very high antitumor activity in several tumor systems. Two of the most active derivatives, namely, 1,4-bis(2-[(2-hydroxyethyl)amino]ethylamino)-9,10-anthracenedione (bisalkylAAD) and 1,4-dihydroxy-5,8-bis(2-[(2-hydroxyethyl)amino]ethylamino)-9,10-anthracenedione (dihydroxybisalkylAAD), which had curative activity in the above-mentioned tumors, were compared in considerable detail. DihydroxybisalkylAAD showed distinct advantages over bisalkylAAD in several tumor systems and is tenfold more potent with respect to effective dose range. This last difference is important for two reasons. First, these aminoanthraquinones are strong and persistent blue dyes and the administration of lower doses would minimize a potential cosmetic drawback of these compounds. Second and most important, iv administration of dose levels of bisalkylAAD which are within the therapeutic dose range on intermittent dose schedules produced convulsions and immediate death. IV administration of dihydroxybisalkylAAD also caused acute toxicity, but, because of its increased potency relative to antitumor activity and delayed toxicity, this acute toxicity was apparent only at doses well above the therapeutic dose range. All of the aminoanthraquinones evaluated, regardless of their activity as antitumor agents in vivo, proved to be potent inhibitors of DNA and RNA synthesis in vitro and bound strongly to DNA as evidenced by deltaTm values (deltaTm = upward shift in DNA melting temperature). Thus, the strong antitumor activity of aminoanthraquinones would appear to be due to some mechanism other than, or in addition to, DNA binding and inhibition of nucleic acid synthesis.


Schedule-dependency assessments of ribonucleoside diphosphate reductase inhibitors when used in combination with platinum compounds plus cyclophosphamide in the treatment of advanced L1210 leukemia. Each of three ribonucleoside diphosphate reductase inhibitors was used as a third drug in combination with selected antitumor platinum (Pt) agents and cyclophosphamide (CY) in the treatment of advanced L1210 leukemia in C57BL/6 x DBA/2 mice. Each was synergistic with the various Pt plus CY combinations but the effect was highly schedule dependent. The collective cure rate was 68% when hydroxyurea (HU) was given as a single injection with Pt plus CY; the cure rate was 15% when HU was administered on a divided-dose schedule with Pt plus CY. The collective cure rate was 53% when guanazole was given as a single injection with Pt plus CY, but was only 8% when it was given on a divided-dose schedule with Pt plus CY. The effect of 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, when used as a third drug with the various Pt plus CY regimens, was not schedule dependent as assessed by the collective cure rate. A therapeutic synergy between CY and each of the three ribonucleoside diphosphate reductase inhibitors was also observed.


Important clinical pharmacologic considerations in the use of methadone in cancer patients. The manufacturer's recommended dose of methadone, which is 2.5-10 mg every 3-4 hours, is excessive. Methadone has an average primary-phase half-life of 14.3 hours and a slower secondary-phase half-life averaging 54.8 hours. Administering the recommended dose of methadone and escalating doses within 2-3 days may lead to very high plasma levels of the drug. In addition, factors such as drug-drug interaction and/or hepatic and renal dysfunction may affect the metabolism of methadone and result in its accumulation. Methadone is an effective analgesic whose pharmacokinetics must be appreciated in order for it to be used safely and effectively. Three cases of methadone overdose are presented.


Therapy for mouse tumors and human tumor xenografts with the antitumor antibiotic AT-125. The antimetabolite antibiotic L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) showed significant antitumor activity against L1210 and P388 mouse leukemias and the M5076 mouse ovarian tumor. Depending on the schedule of administration, increases in lifespan of greater than 100% were observed. Activity was observed after ip, oral, or sc inoculation of AT-125 in mice inoculated with L1210 by the ip route. Lewis lung carcinoma and B16 melanoma were not affected by AT-125. The compound was used to treat human tumor xenografts in athymic (nude) mice. The MX-1 mammary tumor regressed when treated with either 8 or 16 mg/kg/day for 10 days, while a dose of 32 mg/kg was toxic. On an every-4-days x 3 schedule there was a marked slowing of MX-1 tumor growth at 50, 100, and 200 mg/kg. The LX-1 lung tumor xenograft growth was slowed significantly by a dose of 32 mg/kg. Growth of colon tumors, CX-1, CX-2, and CX-3, was not affected by AT-125.


VM-26 as a second drug in the treatment of brain gliomas. Twenty patients previously treated with surgical resection, radiation therapy, and a nitrosourea for malignant gliomas of the brain were given VM-26 as a second chemotherapeutic agent when evidence of tumor progression occurred. Thirty-five percent achieved an arrest or decrease of neurologic symptoms, with a median length of response (arrest and tumor regression) of 22 weeks.


Lipopolysaccharides from Pseudomonas maltophilia: structural studies of the side-chain polysaccharide from strain N.C.T.C. 10257. A lipopolysaccharide has been isolated from Pseudomonas maltophilia N.C.T.C. 10257. Monosaccharide components identified were L-rhamnose, 3-O-methyl-L-xylose, L-xylose, D-glucose, D-mannose, D-galacturonic acid, 2-amino-2-deoxy-galactose, 2-amino-2-deoxyglucose, and a 3-deoxy-2-octulosonic acid. Heptose was absent. In this and other respects, the lipopolysaccharide resembles the corresponding products from Xanthomonas species. Mild hydrolysis of the lipopolysaccharide with acid, followed by chromatography of the water-soluble products on Sephadex G-50, gave a polymeric, "side-chain" fraction containing rhamnose, 3-O-methylxylose, and xylose residues in the molar rations approximately 15:4:1. Methylation analysis, periodate oxidation, Smith degradation, and oxidation with chromium trioxide were the principal methods used in the study of this fraction. The following structure is proposed for the characteristic repeating-unit of the polymer.


Syntheses of O-beta-D-galactopyranosyl-(1 leads to 3)-0-(2-acetamido-2-deoxy-alpha(and -beta)-D-galactopyranosyl)-N-tosyl-L-serine and their interaction with D-galactose-binding lectins. In order to use, as hapten inhibitors against various galactose-binding lectins, the derivatives of O-beta-D-galactopyranosyl-(1 leads to 3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-L-serine, which is the common core structure of sugar chains of most mucins, the synthesis of these compounds was investigated. Koenigs-Knorr condensation of the 4,6-O-benzylidene derivative of O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-N-tosyl-L-serine methyl ester with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide gave O-(2,3,4,L-tetra-O-acetyl-beta-D-galactopyranosyl)-(1 leads to 3)-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-galactopyranosyl)-N-tosyl-L-serine. Deacetylation, followed by acid hydrolysis of the benzylidene group, gave O-beta-D-galactopyranosyl-(1 leads to 3)-O-2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-N-tosyl-L-serine. A beta anomer at the glycoside linkage of the 2-acetamido-2-deoxy-D-galactose residue was also synthesized by the same procedure. Agaricus bisporus (mushroom) hemagglutinin was found to recognize the O-alpha-glycosyl linkage between 2-acetamido-2-deoxy-D-galactose and L-serine, in addition to the O-beta-D-galactopyranosyl-(1 leads to 3)-(2-acetamido-2-deoxy-D-galactose sugar sequence, for which Arachis hypogaea (peanut) and Bauhinia purpurea hemagglutinins were found to be specific. Ricinus communis hemagglutinin is more specific for the O-beta-D-galactopyranosyl-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose (commonly found in serum glycoproteins) than for the O-beta-D-galactopyranosyl-(1 linked to 3)-2-acetamido-2-deoxy-D-galactose sequence. With Wistaria floribunda hemagglutinin, not much difference in the inhibitory activities of these two sugar sequences was observed, and O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-N-tosyl-L-serine was the strongest inhibitor against this lectin.


Chemical structure of the polysaccharide antigen of Eubacterium saburreum, strain O2. The polysaccharide antigen produced by Eubacterium saburreum, strain O2, is composed of (1 leads to 6)-linked beta-D-glycero-D-galacto-heptopyranosyl residues, all of which are substituted with 6-deoxy-alpha-D-altro-heptofuranosyl groups at O-3.


Structural studies of the capsular polysaccharide of Klebsiella type 33. The structure of the capsular polysaccharide from Klebsiella type 33 has been investigated. Methylation analysis, various specific degradations, graded hydrolysis with acid, and n.m.r. spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of pentasaccharide repeating-units having the following structure. (formula, see manual). The D-galactopyranosyl group, with pyruvic acid linked as a ketal to O-3 AND O-4, was degraded on treatment of the fully methylated polysaccharide with strong base. It is proposed that methyl pyruvate is eliminated, in an E2 type of reaction.


Modes of action of intracellular dextranase and three oligoglucanases from Pseudomonas UQM733. The action patterns have been studied of a purified, intracellular dextranase and three intracellular alpha-D-glucosidases from Pseudomonas UQM733 on pure isomalto-oligosaccharides. The glucosidases have optimal activity on isomaltotetraose and are therefore classified as oligoglucanases. They have been used to determine the structure of two branched isomalto-oligosaccharides obtained by enzymic degradation of dextran.


Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B. Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.


An examination of some basic assumptions of DNA distribution analysis using biological data. An examination is made of how the observed DNA distributions spread from their original biological distributions. Using distributions from testicular and hepatic tissue we find that the current assumptions for DNA distribution analysis need reexamination and suggest how this might be done.


Labelling index of human squamous cell carcinomas. Comparison of in vivo and in vitro labelling methods. In vivo and in vitro labelling has been compared in sixteen human solid squamous cell carcinomas (ENT). The median in vivo/in vitro LI ratio was 1-2, but for two-thirds of the patients it was only 1-1, suggesting a slight LI underestimation in vitro. Two factors can possibly explain the divergences: heterogeneity from one biopsy to another in the same tumour, and lower mean grain count in the deep cell layers of the in vitro labelled tumour samples. Therefore, the fact we did not find a good agreement between in vivo and in vitro data for one-third of the tumours points out that one must be cautious in considering in vitro LI as a valid result for a given patient. However, even if the in vitro LI leads to a certain underestimate, it can provide useful data.


Effect of an inhibiting factor isolated from rat liver on DNA polymerases in regenerating rat liver. We partially purified an inhibitory factor (LIFE), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G1--S transition, and reduced in vivo [3H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented alpha DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on alpha DNA polymerase was detected. LIF did not affect beta DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.


The relationship between stem cell seeding efficiency and position in cell cycle. The seeding efficiency of colony-forming cells from normal, regenerating and velocity-sedimented cycling and non-cycling narrow preparations was compared. Colony-forming cells in cycle were found to exhibit a 50% reduction in splenic seeding when compared to normal marrow or sedimented non-cycling cells. The results of this study indicate that the spleen colony assay underestimates the total number of colony-forming cells by a fraction which is directly related to the number of cells in cycle.


Cell population kinetic profile of the mouse thymus, and the changes induced by prednisolone. The cell population kinetic parameters of the thymus in BALB/c mice have been estimated using stathmokinetic and [3H]TdR techniques in both control animals and animals treated with prednisolone. FLM data were analysed by computer using the Gilbert program. The study showed that prednisolone had an inhibitory effect mainly in the DNA synthesis phase and in G1. Stathmokinetic data also showed a decrease in the cell birth rate and an increase in the apparent cell cycle time (or potential doubling time) after treatment. The labelling index, the mitotic index and the growth fraction were also decreased. The study also shows a good agreement between the data obtained by stathmokinetic and [3H]TdR techniques.


Relative toxicities of particulate and soluble forms of beryllium to a rat liver parenchymal cell line in culture and possible mechanisms of uptake. The relative toxicities of particulate beryllium phosphate, soluble beryllium sulphate and a beryllium sulphosalicylate complex to a rat liver parencymal derived cell line have been examined in culture. Due to the propensity of beryllium salts to form beryllium phosphate in solution the incubation medium used was free of inorganic phosphate. Cell death measured by the loss of cellular lactate dehydrogenase into the medium can be produced within 76 h from beryllium phosphate and beryllium sulphosalicylate or 48 h from beryllium sulphate provided the cells have, irrespective of the form of added beryllium, taken up a minimum of 2--5 nmol Be/10(6) cells. Whilst beryllium phosphate was readily taken up as a particle, beryllium complexed with excess sulphosalicylate was not so markedly accumulated by the cells except possibly by formation of small amounts of beryllium phosphate in the medium as a result of inorganic phosphate lost from the cells. The extent of beryllium uptake from beryllium sulphate quantitatively most resembled that observed for beryllium phosphate but was largely independent of beryllium phosphate formation in the medium and not accompanied by the uptake of the SO42- anion. However, the accumulation of beryllium derived from beryllium sulphate did appear to be associated with the production of a sedimentable from believed most probably to be colloidal beryllium hydroxide. The uptake of all forms of beryllium was temperature sensitive and metabolic inhibitor studies and treatment of the cells with trypsin or neuraminidase supported the view that the distinct behaviour of beryllium derived from beryllium sulphate may be related to the enhanced toxicity of this form both under the conditions used and when administered to experimental animals.


Mode of action of formamidine pesticides: an evaluation of monoamine oxidase as the target. The ability of formamidine pesticide, chlordimeform (N'-(4-chloro-o-toyl)-N,N-dimethylformamidine) (CDM), and several of its major metabolites to inhibit monoamine oxidase (MAO) in mouse tissues in vitro and in vivo was examined, and related to the hypothesis that inhibition of MAO is responsible for the lethal effects of CDM. CDM was a readily reversible inhibitor of MAO of medium potency as were most of its metabolites. However, the hydrolysis product, N-formyl-4-chloro-o-toludine (CT) was a significantly more potent reversible inhibitor. A comparison of MAO from brain, liver, and intestine showed no marked variations in their sensitivity to these inhibitors. Greater inhibitory potency was found using Type A substrates (5-hydroxytryptamine) than Type B substrates (beta-phenylethylamine). The activity of MAO in vivo after pretreatment of mice with CDM or its metabolites was assessed in liver and intestine by measuring the amount of [14C] tryptamine which still survived 5 min after an intraperitoneal injection. Established inhibitors of MAO gave appropriate results with this method. CDM also increased tryptamine recoveries but only at does which caused mortality, and then to a lesser extent than MAO inhibitors such as tranylcypromine, pheniprazine, and harmaline at sub lethal doses. For this reason, and in view of the lack of correlation of toxicity to MAO-inhibitory potency among CDM and its metabolites, and because the symptoms of poisoning are inappropriate, it is concluded that MAO inhibition is not an important factor in the acute lethality of CDM.


Response of two strains of L5178Y cells to cis-dichlorobis(cyclopentylamine)platinum(II). I. Cross-sensitivity to cis-PAD and UV light. The response to cis-dichlorobis(cyclopentylamine)platinum(II) (cis-PAD) an antitumour platinum complex, was studied in two strains of murine lymphoma L5178Y cross-sensitive to X-rays and UV light. Dose-survival relationship, DNA synthesis formation of chromatid aberrations, progression through the cell cycle, and growth and viability changes after 1 h cis-PA; treatment at 37 degree C were examined and compared with the effects of X-rays and UV light. In both strains, cis-PAD caused immediate inhibition of progression through the cell cycle, reduced rate of DNA synthesis, delayed appearance of chromatid aberrations, and delayed death; however, there is a marked difference in sensitivity to cis-PAD between L5178Y-S strain (D0 approx. 5.8 microgram/ml) and L5178Y-R strain (D0 approx. 2.5 microgram/ml). In both strains a close resemblance was found between dose-survival relationships after cis-PAD and UV light treatment, respectively.


Response of two strains of L5178Y cells to cis-dichlorobis(cyclopentylamine)platinum(II). II. Differential effects of caffeine. Two strains of L5178Y murine lymphoma, inversely cross-sensitive to X-rays and UV light, were shown previously to respond to treatment with an antitumour platinum complex, cis-dichlorobis(cyclopentylamine)-platinum(II) (cis-PAD), in a similar manner as to UV. Enhancement of chromosomal damage and potentiation of lethal effect of cis-PAD by 0.75 mM caffeine were found in cis-PAD and UV light-resistant L5178Y-S strain but not in cis-PAD and UV light-sensitive L5178Y-R strain. These results suggest that the extreme sensitivity of L5178Y-R strain to cis-PAD and UV light is caused to some extent by deficiency in a caffeine-sensitive post-replication repair system.


Effect of free fatty acid anion on the in vitro binding of 1-anilino-8-naphthalene sulfonate to cytoplasmic proteins from rat liver. The influence of oleate ion, a free fatty acid anion, on the binding characteristics of 1-anilino-8-naphthalene sulfonate (ANS) with the cytoplasmic proteins (Y and Z) from rat liver has been examined using fluorescence spectroscopy. ANS binds strongly with both ligandin (Y) and Z protein at a single binding site with dissociation constants of 0.6 and 1.4 micron respectively. Increasing concentrations of oleate ion decreased the ANS binding with either protein by competing with the ANS binding site. Relative binding constant of oleate ion for the hepatic ligandin or Z protein was about 2 micron as determined from the competitive inhibition of ANS binding. These results suggest that variations in the hepatic cytoplasmic free fatty acid concentration may be important in regulating the capacity of Y and Z proteins to transport other organic anions.


Differential cytotoxic activity of potassium dichromate on nucleoside uptake in BHK fibroblasts. In cultures of hamster fibroblasts (BHK cell line) treated with potassium dichromate (K2Cr2O7) nucleic acid and protein syntheses are differentially inhibited, and nucleoside uptake into the intracellular pool is characterized by a stimulation phase followed by an inhibition phase. Different patterns are observed for the uptake of each ribo- and deoxyribonucleoside, pyrimidine nucleoside (particularly deoxycytidine) uptake reaching the highest stimulation level. Kinetics of thymidine and deoxycytidine initial uptake at different exogenous nucleoside concentrations show that K2Cr2O7 affects both simple and facilitated diffusion of nucleosides. The time course of thymidine and deoxycytidine pool saturation suggests however that the effects of K2Cr2O7 on plasma membrane permeability are partially counterbalanced by modifications of pool size deriving from the concomitant alteration of steps of nucleoside metabolism separate from nucleoside uptake.


Biotransformation of chloroform by rat and human liver microsomes; in vitro effect on some enzyme activities and mechanism of irreversible binding to macromolecules. The effects of chloroform on some rat microsomal enzyme activities were studied in vitro. Maximum inhibition of oxygen consumption, NADPH oxidase and NADPH-cytochrome c reductase was observed at 0.5 mM chloroform; prior metabolization of CHCl3 by microsomal monooxygenases increased inhibition by about 50% at 0.2-0.5 mM chloroform. Higher concentrations produced a paradoxical reversal of inhibition, whereas p-nitroanisole demethylase was steadily inhibited by about 50% up to 10 mM chloroform. Irreversible binding of 14CHCl3 was confirmed to depend on chloroform metabolization by monooxygenases. The increased irreversible binding due to phenobarbital induction is accompanied by a diminished affinity towards chloroform as shown by increased KM of irreversible binding, and a higher spectral dissociation constant KS. Aminoacids with nucleophilic functions (histidine, cysteine) partially prevented the irreversible binding of chloroform metabolites to microsomes; non-volatile radioactive derivatives were recovered in trichloracetic acid supernatants when microsomes were incubated with cysteine, but not with histidine. Phosgene has been demonstrated as a biological metabolite of chloroform: its possible reactions with nucleophilic groups of macromolecules, water and added aminoacids partly explain these experimental data. Similar results were obtained with human microsomes, showing that chloroform hepatotoxicity in man could involve the same mechanisms.


Fate of juvenile hormone in mammalian cell culture. The metabolism of juvenile hormone (JH) I has been examined in fetal mouse liver cells maintained in culture. Diffusion of the hormone into the cells appears to be passive. The hormone is metabolized essentially to organic-soluble metabolites (diol ester, diol acid and acid) by the action of epoxide hydrase and carboxylesterases. Conjugative reactions play a minor role, less than 3% of the hormone being excreted as conjugates (glucuronides, sulfates and mercapturic acid). About 0.8% of the cellular radioactivity is bound to macromolecules, mainly those of nuclear and mitochondrial origin. Metyrapone and SKF 525-A inhibit covalent binding of the hormone to cytoplasmic macromolecules, which suggests participation of the cytochrome P-450 system in covalent binding of the hormone.


A class of strong inhibitors of microsomal monooxygenases: the ellipticines. Ellipticine (E) and its 9-hydroxy derivative inhibit strongly various liver monooxygenase activities mediated by microsomes from control and phenobarbital (PB), benzo[alpha]pyrene (BP) or Aroclor 1254 (Aroclor)-pretreated rats. The inhibition constants, Ki, are remarkably low, and often smaller than 1 micron, particularly in the case of microsomes containing cytochrome P-448. The inhibitory potency (I50) of 9-hydroxyellipticine (9-OHE) is larger (about ten-fold) than the one of classical inhibitors (metyrapone or 7,8-benzoflavone (7,8-BF)), whatever the activities studied and the induction of microsomes. Differences exist between the mechanisms of inhibition according to the form of cytochrome P-450 present in microsomes of differently pretreated rats; whichever the activities studied, one observes: (a) a competitive inhibition towards the activity of non-induced or PB-induced microsomes and (b) a non-competitive inhibition towards the activity of Aroclor or BP-induced microsomes, at variance with 7,8-BF. These results are in good agreement with the interaction properties of the ellipticines with microsomal cytochromes P-450.


The effect of cephaloridine on the stability of rat kidney lysosomes. The administration of cephaloridine to rats caused a decrease in the excretion of acid phosphatase into the urine. The antibiotic itself had no effect on urinary acid phosphatase and inhibitors or proteolytic enzymes were not present in the urine from treated rats. Cephaloridine may therefore be stabilizing the lysosomal membrane in vivo and experiments with isolated lysosomes confirm this hypothesis. The lysosomal integrity was followed by measuring the acid phosphatase activity and the light scattering properties of the particles. A good correlation was obtained between these parameters in the case of thermal disruption and progesterone induced lysis of the lysosomes and low concentrations of cephaloridine (0.1-1.0 mmol/1) protected the lysosomes against this form of damage.


Enhanced inhibition of both cellular protein synthesis and malate dehydrogenase by aged aquoplatinum(II) complexes. Previous studies have shown cis-diamminedichloroplatinum(II) (Cis) an effective anti-tumour agent in man and animals. Evidence is presented here that formation of aquo complexes of this platinum derivative will significantly enhance its inhibitory properties with respect to two separate biochemical functions, namely inhibition of protein synthesis in hamster medulloblastoma cells and in inhibiting the activity of L-malate dehydrogenase (MDH) in a cell free system. Inhibition of cell protein synthesis rises from 8% using freshly dissolved drug to 30% when aged solutions of drug are employed at an inhibitor concentration of 0.1 mM. The inhibitory enhancement seen using purified malic dehydrogenase increases from 16% (fresh) to 57% (aged) at an inhibitor concentration of 1 mM.


Molecular structural effects involved in the interaction of anthracyclines with DNA. Changes in DNA binding ability of daunomycin following structural modifications in the aglycone moiety have been studied by the fluorescence quenching method and by thermal denaturation of the complex. Removal of the methoxyl group at position 4 leads to a slightly stronger binding. Changes in the position of the glycosidic linkage result in a markedly weaker binding. Removal of the hydroxyl group at position 9, with the concomitant formation of a 9,10-anhydro derivative, decreases the binding ability. Methylation of hydroxyl groups at C-6 and C-11 leads to an inactive derivative and makes the binding affinity disappear almost completely. Structure-activity correlations for the DNA binding reaction deduced from these studies are in agreement with earlier findings that relate to the biological activity and confirm the general picture of the binding mechanism.


Studies on lung tumours. IV. Correlation between [3H]thymidine labelling of lung and liver cells and tumour formation in GRS/A and C3Hf/A male mice following administration of dimethylnitrosamine. Male mice of the inbred strain GRS/A are highly susceptible to lung tumour but refractory to liver tumour formation, whereas the opposite relation holds for C3Hf/A male mice. Liver and lung cells of these 2 mouse strains were studied autoradiographically after intraperitoneal injection of [3H]dimethylnitrosamine (DMN) and of [3H]thymidine at days 1--14 after administration of unlabelled DMN. Corresponding cell types in the lungs or livers of these 2 mouse strains bound similar amount of [3H]DMN. Among the various types of lung cells only the alveolar Type II cells, from which the lung adenomas derive, showed a strain-specific difference in [3H]thymidine labelling indices, much more cells becoming labelled in the case of the GRS/A than of the C3Hf/A strain at days 3--7 after carcinogen administration. Opposite thymidine labelling indices were exhibited by the parenchymal liver cells of the 2 strains, with C3Hf/A now showing a greater response than did GRS/A males. Thus thymidine-labelling and tumourigenic responses of target lung and liver cells to carcinogen in the 2 strains coincided. Sulphur dioxide and carbon tetrachloride mimicked the effects of DMN on the thymidine labelling indices of, respectively, the lung alveolar Type II and the thymidine labelling indices of, respectively, the lung alveolar Type II and the liver parenchymal cells of the 2 strains. The nature of the differential effect of carcinogen on the [3H]thymidine labelling of the cells and the correlation of these patterns with susceptibility to tumour formation, are briefly discussed.


The interactions of triethyltin with rat glutathione-S-transferases A, B and C. Enzyme-inhibition and equilibrium-dialysis studies. Purified glutathione(GSH)-S-transferases A, B and C from rat liver are inhibited by triethyltin (SnEt3). With 1-chloro-2,4-dinitro benzene (CDNB) as the limiting substrate the inhibition is competitive in each case. At a GSH concentration of 5 . 10(-3) M the inhibition constants for transferases A and C at 25 degrees C are similar and very low, 3.2 . 10(-8) M and 5.6 . 10(-8) M respectively, whereas for transferase B the inhibition constant is 3.5 . 10(-5) M. Equilibrium-dialysis experiments carried out at 4 degrees C in the absence of GSH give apparent dissociation constants of 7.1 . 10(-4) M and 3.4 . 10(-4) M for transferases A and B respectively, but if 5 . 10(-3) M glutathione is included in the dialysis solutions these values fall to 2.0 . 10(-7) M and 2.6 . 10(-5) M, which are within an order of magnitude of the kinetic Ki-values. Chromatographic experiments with Sephadex G-10 show that GSH and SnEt3 interact in aqueous solution under the conditions of the enzyme-kinetic and equilibrium-dialysis experiments. It is suggested that the inhibited enzymes are in the form of ternary complexes, enzyme-GSH-SnEt3, in which GSH and SnEt3 may or may not interact directly; or are possibly quaternary complexes, enzyme-(GSH)2-SnEt3. SnEt3 could be valuable as a selective inhibitor of transferases A and C in mixtures of the three transferases.


Zonal changes in the rat liver after chronic administration of phenobarbitone in ultrastructural, morphometric and biochemical correlation. Alterations in the liver of rats subjected to 24 days of continuous administration of phenobarbitone have been supplied bu subcellular fractionation, conventional electron microscopy and morphometric analysis. The increase in wet weight of the liver was found to result from a combination of cellular hypertrophy, hyperplasia and an enlarged hepatic blood space. In the centrilobular zone all the hepatocytes underwent a substantial proliferation of total ER, became enlarged and had an increased blood supply. However, in the periportal zone phenobarbitone caused changes in only 45% of the hepatocytes, the remainder being apparently resistent or tardy. An overall dramatic increase in hepatic RER was both measured and observed but the response involved hepatocytes in which the RER had proliferated as well as those which were depleted of RER or had stacks and cisternae that were severely shortened and dispersed. These alterations are discussed in relation to changes in RER after administration of agents causing hepatonecrosis. Possible reasons for the inability of other workers to detect a phenobarbitone-induced increase in RER are also put forward. After subcellular fractionation and corection for centrifugation losses into the 9500 g pellet, using the microsomal marker cytochrome P-450, phenobarbitone-induced increase in total ER was substantially less than that found by morphometric analysis. This indicates that during the preparation of microsomes a substantial proportion of intracellular membranes, having different metabolic and synthetic properties to those finally isolated, are discarded and emphasizes the need to exercise care when using microsomal preparations.


The in vitro and in vivo reaction at the N7-position of guanine of the ultimate carcinogen derived from benzolalpyrene. The previously reported reaction at N2- and N7- of guanine following addition of 7 alpha,8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) to an aqueous solution of DNA has been studied in more detail. The extent of reaction and the relative yields of N2- and N7-products was measured over the range of pH 4--7. The depurination following reaction at the N7-position of guanine was found to have a half-life of 3 h. Reaction of the isomeric 7 alpha,8 beta-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (syn-BPDE) with DNA gave the expected N2- and no N7-guanine product. When either benzo[a]pyrene or anti-BPDE was added to mouse embryo or Chinese hamster V79 cells respectively, a major N2-guanine product and a very minor adenine product were isolated from the DNA, but no N7-guanine product was detected.


The binding of metabolites formed from aminostilbene derivatives to nucleic acids in the liver of rats. Carcinogenic trans-4-dimethylaminostilbene (trans-DAS) and trans-4-acetylaminostilbene (trans-AAS) as well as inactive cis-DAS and DABB were highly and specifically labeled with tritium and administered orally to female Wistar rats. Covalent binding to liver rRNA and DNA was measured and found to be higher for the carcinogenic compounds. Digests from these nucleic acids were chromatographed on Sephadex LH-20 and 16 different nucleoside adducts were characterised by their retention volumes. Labeled trans-DAS was administered in doses ranging from 0.025--250 mumol/kg. Binding to nucleic acids was directly proportional to the dose at low doses (0.025--2.5 mumol/kg) and less than proportional at higher doses (25--250 mumol/kg). The pattern of nucleoside adducts remained practically constant over the wide range of doses. A pharmacokinetically determined threshold of metabolic activation thus could not be demonstrated for this compound. A modified procedure is described to simultaneously isolate pure liver rRNA and DNA from nonfasted rats in high yields.


Sympathetic control of major coronary artery diameter in the dog. The diameter of a major coronary artery, the ramus interventricularis ventralis (RIV), was measured in dogs with arrested hearts perfused by an extracorporeal circulation. The resting diastolic diameter was 1.78 +/- 0.07 mm (mean +/- SE) at a diastolic pressure of 74.2 +/- 3.4 mm Hg. Bilateral supramaximal stimulation of fibers leaving the cranial pole of the stellate ganglion decreased the diameter by 71.2 +/- 8.9 micrometer, i.e., 4.0 +/- 0.5% of the resting diameter. Stimulation of the left stellate ganglion contributed 59.8 +/- 5.7% of the maximum response; that of the right contributed 40.3 +/- 5.5%. Stimulation of the thoracic ganglia (T2-4) resulted in a 1.2 +/- 0.4% decrease in coronary vessel diameter. RIV failed to respond to bilateral caudal cervical ganglion stimulation. After iv administration of phentolamine, 1-2 mg/kg, no response to sympathetic stimulation could be elicited. Therefore, it appears that alpha-receptors are activated by the release of the sympathetic neurotransmitter to sympathetic stimulation and that beta-receptors are not involved in the response of RIV to sympathetic stimulation.


Myocardial infarct size and ventricular function in rats. To define the relationship between infarct size and ventricular performance, we performed hemodynamic studies in rats 21 days after left coronary artery occlusion. Ventricular performance was assessed under ether anesthesia by measurements of baseline hemodynamics and stressed performance as determined by the peak cardiac output and stroke volume obtained during intravenous volume loading and by the peak left ventricular developed pressure obtained during occlusion of the ascending aorta. Infarct size was determined by planimetry of the endocardial circumference of each of four histological slices of the left ventricle. Rats with small (4-30%) myocardial infarctions had no discernible impairment in either baseline hemodynamics or peak indices of pumping and pressure-generating ability when compared to the sham-operated, noninfarcted rats. Rats with moderate (31-46%) infarctions had normal baseline hemodynamics but reduced peak flow indices and developed pressure. Rats with infarctions greater than 46% had congestive heart failure, with elevated filling pressures, reduced cardiac output, and a minimal capacity to respond to pre- and after load stresses. The entire spectrum of postinfarction ventricular function was observed, from no detectable impairment to congestive failure. In this model of histologically healed myocardial infarction, the impairment of left ventricular function was directly related to the loss of myocardium.


Influence of cardiac fiber orientation on wavefront voltage, conduction velocity, and tissue resistivity in the dog. When the canine epicardium is stimulated, the spread of epicardial excitation is 2.4 times faster parallel to the long axes of the cardiac fibers than perpendicular to them. Likewise, gross tissue resistivity is lower parallel to fibers by a factor of 3.2, and the voltage across the depolarization wave is approximately three times as great in the longitudinal direction. Equations are presented which relate these variables. Theoretical considerations confirm the experimental finding that the potentials around a wave of depolarization cannot be accounted for by the conventional hypothesis that the wavefront is a uniform double-layer current source.


Cardiovascular and pulmonary effects of thromboxane B2 in the dog. The hemodynamic properties of thromboxane B2 (TxB2), a product of prostaglandin endoperoxide metabolism, have not been thoroughly described. TxB2 is a bronchoconstrictor, but its effects on the systemic circulation and circulating platelets are unknown. Its precursor, thromboxane A2(TxA2), is a potent vasoconstrictor as well as a platelet-aggregating agent. Using intact anesthetized dogs, we investigated the effects of TxB2 on pulmonary artery pressure (PAP), airway pressure (AP), systemic arterial pressure (SAP), and myocardial contractility (MC). Vascular responses were evaluated in relation to changes in platelet population and aggregability. Intravenous TxB2 (25 and 50 micrograms/kg) increased AP (mean 62% and 69%) and PAP (50% and 86%), respectively, whereas SAP and MC responses were inconsistent. Left ventricular injections (25 micrograms/kg) also increased AP (36%) and PAP responses were inconsistent. Left ventricular injections (25 micrograms/kg) also increased AP (36%) and PAP (36%). Intraventricular administration of TxB2 produced a consistent elevation of SAP (10%) with a concomitant fall in MC (11%). These vascular responses were not consistent with alterations in platelet number or aggregability. A tachyphylactic response to TxB2 developed in AP and PAP at both dose levels and with both routes of administration. Intravenous and intraventricular TxB2 (25 micrograms/kg) produced a parallel decreasing response in PAP, suggesting the possible saturation of TxB2 binding sites or the depletion of a catabolic enzyme in the lung.


The role of adenosine in prolonged vasodilation following flow-restricted exercise of canine skeletal muscle. A period of prolonged vasodilation follows flow-restricted exercise of skeletal muscle. We tested the hypothesis that adenosine participates in mediating this vascular response. Vascularly isolated, anterior calf muscles of anesthetized dogs were stimulated to contract at a rate of 4 twitches/sec. Blood flow was held constant at 12.5 +/- 1.3 ml/min per 100 g which was about 14% of the expected free flow for this exercise level. Skeletal muscle tissue adenosine was measured with the an enzymatic, spectophotometric assay of trichloroacetic acid extracts of congruent to 50 mg biopsy samples. Tissue adenosine rose from 2.30 +/- 0.90 nmol/g in resting muscle to 22.5 +/- 5.8 nmol/g by the end of the 22-minute exercise. Following exercise, tissue adenosine fell toward its baseline value with a time course very similar to the early portion of the return of skeletal muscle vascular resistance to its control level. Thus, skeletal muscle adenosine content (1) increases to a sufficient magnitude and (2) falls with an appropriate time course to be at least partly responsible for the early portion of prolonged vasodilation seen after flow-restricted exercise of skeletal muscle.


The role of potassium in the metabolic control of coronary vascular resistance of the dog. We tested the hypothesis that potassium ion (K+) is involved in the local control of the coronary circulation. The left coronary artery was perfused at constant flow in closed-chest, anesthetized dogs. Step increases in heart rate caused transient (six dogs) or sustained (three dogs) increases in coronary sinus plasma [K+] averaging 0.53 mEq/liter. When the effects of vascular transit delay were accounted for, we found that [K+] changes preceded the vasodilation seen with increased heart rate. We used a mathematical model to calculate changes in interstitial [K+] from arterial and venous [K+] and K+ release rate. The magnitude of the changes in interstitial [K+] appeared to be sufficient to account for a considerable portion but not all of the initial changes in coronary vascular resistance associated with increased heart rate. Thus potassium seems to be involved at least transiently, and, in three of nine dogs, for a more sustained period, in heart rate-induced coronary vasodilation. Cessations (for 15 seconds) of coronary blood flow resulted in transient postischemic increases of coronary sinus [K+] averaging 0.55 mEq/liter. In this case, correction for vascular transit disclosed that the recovery of [K+] preceded the return of vascular tone to baseline for only the second half of the recovery, implying only a limited role for potassium in this response. Potassium appears to play a significant but transient role in the local control of the coronary circulation.


High energy phosphate stores and lactate levels in different layers of the canine left ventricle during reactive hyperemia. To study postischemic recovery of myocardial energy metabolism in relation to the reactive hyperemic response, the tissue levels of creatine phosphate (CP), adenosine triphosphate (ATP), and lactate were estimated in the outer, middle, and inner layers of the left ventricle before, during, and at several times after a 20-second bilateral coronary arterial occlusion in the open-chest dog. The reactive hyperemic response was characterized by monitoring blood flow in the cannulated coronary sinus in a separate group of dogs. Substantial changes in myocardial CP and lactate, but not ATP, were produced by the occlusion and reciprocal transmural gradients in CP and lactate occurred such that CP was lowest and lactate was highest in the inner layer. Recovery of high energy phosphate stores (CP + ATP) occurred long before blood flow returned to the preocclusion level, this being achieved in two of the three layers after completion of only 29% of the reactive hyperemic response when the blood flow debt repayment was 151%. Lactate returned to control levels during the response, but recovery times in the different layers were delayed compared to those for the high energy phosphate stores. A transmural lactate gradient was maintained as the regional levels declined, and recovery times were different for each of the three layers, being longest in the inner layer. The results suggest that during the reactive hyperemic response (1) kinetically different processes are involved in the repletion of energy stores and the removal of anaerobically produced metabolites, and (2) metabolic vasodilation of coronary vessels probably is more pronounced and sustained longer in the inner than in the outer ventricular layer.


Effects of K+ and K+-induced polarization on (dV/dt)max, threshold potential, and membrane input resistance in guinea pig and cat ventricular myocardium. We studied the non-membrane potential-dependent effect of K+ on (dV/dt)max and threshold potential in guinea pig and cat ventricular myocardium. Membrane potential (MP) was changed uniformly in segments (length less than or equal to 1.0 mm) of papillary muscles by applying extracellular polarizing current pulses across a single sucrose gap. Control [K+]o was 5.4 mM and test [K+]o values were 2.0, 10.0, 11.5, 13.0, 16.2, 20, 22, and 24.0 mM. Each muscle was studied under four conditions: (1) control [K+]o and unaltered (control level) resting MP (Em); (2) one of the test [K+]o values and the unaltered (test level) Em; (3) the same test [K+]o and Em held at the control level; (4) control [K+]o and Em held at the test level. At all [K+]o greater than or equal to 11.5 mM, (dV/dt)max showed a decrease significantly (P less than 0.01) greater than the corresponding MP-dependent decrease in both guinea pig and cat myocardium. This non-MP-dependent decrease averaged 7.5% at 11.5 mM, 26.5% at 13.0 mM, 37.2% at 16.2 mM, and 22.7% at 20.0 mM. At [K+]o greater than or equal to 20.0 mM, (dV/dt)max was predominantly slow-channel-dependent; it was increased by hyperpolarization to -110 mV at [K+]o = 20 and 22 mM but not at [K+]o = 24mM. Threshold potential became progressively less negative with increasing [K+]o, but this effect was dependent only on MP. The membrane input resistance (rm) was determined by two opposing factors: at a given [K+]o, rm increased with depolarization; and at a given MP, rm decreased with increasing [K+]o. Our study shows that non-MP-dependent depression of (dV/dt)max in the ventricular myocardium occurs at [K+]o concentrations that may be encountered in vivo.


Stimulation of renin by acute selective chloride depletion in the rat. To determine whether acute chloride depletion per se stimulates renin, we produced selective chloride depletion without sodium depletion in rats by peritoneal dialysis (PD) against 0.15 M NaHCO3 or 0.15 M NaNO3. Control rats were dialyzed against 0.15 M NaCl. Plasma renin activity (PRA) was measured before (PRA1) and 105 minutes after (PRA2) PD. Plasma volume was expanded after PD by infusion of salt-free albumin and was measured immediately after PRA2 by [131I]albumin. In experiment 1, rats were prepared on a normal diet. PRA2 (7.0 +/- 1.0 ng/ml per hr, mean +/- SEM) was increased (P less than 0.05) over PRA1 (4.7 +/- 0.7 ng/ml per hr) in Cl-depleted but not in control rats (PRA1 = 5.3 +/- 0.7, PRA2 = 6.1 +/- 0.7, P = NS). In experiment 2, to produce greater chloride depletion, all rats were prepared for 2 weeks on a low salt diet. PRA2 (47 +/- 5 ng/ml per hr) was increased as compared to PRA1 (24 +/- 2 ng/ml per hr, P less than 0.005) in the Cl-depleted group but not in the control group (PRA1 = 24 +/- 3, PRA2 = 27 +/- 6 ng/ml per hr, P = NS). Serum potassium and final plasma volume were slightly but not significantly lower than controls in these Cl-depleted rats. To exclude an additive effect of these two stimuli for renin, in experiment 2a we infused chloride-depleted rats with three times as much albumin as controls and with KHCO3, 100 mEq/liter. Despite volume expansion and potassium loading, PRA2 (41 +/- 6 ng/ml per hr) was significantly elevated as compared to PRA1 (25 +/- 4 ng/ml per hr, P less than 0.01). Since acute metabolic alkalosis also was present in all Cl-depleted renin-stimulated rats, an additional group (2b) was dialyzed against 0.15 M NaNO3; final plasma arterial pH (7.43) was not different from controls (7.42). Nevertheless, PRA2 levels again were higher (36 +/- 6 ng/ml per hr, P less than 0.05) as compared to PRA1 (23 +/- 4 ng/ml per hr). In all experiments, arterial blood pressure, glomerular filtration rate, and filtered sodium load were not different. Free water reabsorption was lower in Cl-depleted than in control rats. We conclude that acute selective chloride depletion per se is a potent stimulus for renin release.


Potential arrhythmogenic electrophysiological derangements in canine Purkinje fibers induced by lysophosphoglycerides. We have recently detected accumulation of lysophosphoglycerides, catabolites of phospholipids, in ischemic myocardium early after coronary occlusion. In the present study we delineated effects of selected concentrations of albumin-bound lysophosphatidyl choline (LPC) comparable to those accompanying ischemia in vivo on action potentials of isolated canine Purkinje fibers. Lysophosphoglycerides induced concentration-dependent (0.75-3.0 mM) decreases in resting membrane potential, overshoot of phase 0, maximal velocity of upstroke (Vmax) of phase 0, and action potential duration. The highest concentrations (2.0-3.0 mM) induced fractionation of the action potential into several components, unresponsiveness to external stimulation, and enhanced automaticity at normal and reduced membrane potentials. LPC induced a rightward shift in the membrane response curve, a 40-fold prolongation of conduction time, and an increase in the ratio of effective refractory period to action potential duration such that the effective refractory period persisted beyond action potential duration, resulting in postrepolarization refractoriness. These electrophysiological alterations were entirely reversible after 70 minutes of perfusion without LPC, with the exception of a persistent depression in the Vmax of phase 0. Lysophosphatidyl ethanolamine (LPE) elicited alterations in action potentials indentical to those elicited by LPC. Furthermore, LPC (3.0 mM) induced comparable alterations in action potentials recorded from isolated rabbit papillary muscles. Since lysophospholipids accumulate early after myocardial ischemia, and since concentrations equivalent to those occurring in vivo induce electrophysiological alterations resembling those seen in ischemic myocardium in vivo, lysophosphoglycerides may be of major importance as biochemical mediators of malignant dysrhythmia induced by ischemia.


Protracted ventricular tachcardia induced by premature stimulation of the canine heart after coronary artery occlusion and reperfusion. The effects of premature ventricular stimuli were studied in two groups of dogs with infarcts, one group subjected to permanent occlusion of the left anterior descending coronary artery and the other to temporary occlusion for 2 hours. In dogs with permanent occlusion, spontaneous ventricular arrhythmias occurred after 3-6 hours. In 13 dogs with temporary occlusion, ventricular arrhythmias occurred immediately after reperfusion and then persisted. In five dogs with temporary occlusion, ventricular arrhythmias did not occur spontaneously until 13-15 hours after occlusion. On days 2-9 after surgery, after sinus rhythm had returned, the ventricles of each awake dog were stimulated. After permanent occlusion, premature stimuli occurring on the T wave usually induced from one to 10 repetitive responses on days 2-4. Protracted ventricular tachycardia (lasting greater than 10 seconds) was induced in only two of 10 dogs. The response to premature stimuli was similar after temporary occlusion when ventricular arrhythmias did not occur spontaneously until 13-15 hours after occlusion. Protracted tachycardia was not induced. In the dogs with temporary occlusion, which initially had continuous arrhythmias, premature stimuli occurring on the T wave on days 3-5 after surgery induced both repetitive responses and protracted ventricular tachycardia. Stimuli applied to the ventricles during tachycardia terminated it. Histological studies on all infarcts showed that, after permanent occlusion, necrosis was uniform; after temporary occlusion, viable myocardium survived in the necrotic region. These salvaged myocardial fibers may provide reentrant pathways, causing long-lasting tachycardia.


Acceleration of idioventricular rhythms by histamine in guinea pig heart: mediation by H2 receptors. To evaluate the ability of histamine to induce ventricular arrhythmias, we studied the effects of histamine on ventricular rhythmicity in the isolated guinea pig heart with complete atrioventricular conduction block. As a function of dose (0.1-30 microgram), histamine enhanced the idioventricular rate by increasing the rate of firing of the original pacemaker and also by causing the sudden appearance of faster idioventricular rhythms that coincided with changes in pacemaker site. Anaphylaxis in the isolated guinea pig heart with complete atrioventricular conduction block caused histamine release and acceleration of idioventricular rate. The effects of histamine on idioventricular rhythmicity were not attenuated by the histamine H1 receptor antagonist chlorpheniramine, but were antagonized by the H2 receptor antagonist cimetidine. Moreover, the selective H2 agonist 4-methylhistamine (4MeH) accelerated the idioventricular rate, whereas 2-(2-thiazolyl) ethylamine (ThEA), at doses selective for H1 receptor activation, did not. The effects of histamine on idioventricular rhythmicity were not modified by the beta-adrenergic blocker pindolol. The mechanism by which histamine increases idioventricular rate probably involves two components: (1) an enhancement in automaticity of the original pacemaker, and (2) the induction of faster rhythms via reentry and/or afterdepolarizations. Whatever the mechanism, both components of the ventricular chronotropic action of histamine appear to involve exclusively histamine receptors of the H2 type. Thus, our results suggest that H2 receptor antagonists may have a role as specific antiarrhythmic agents in the treatment of cardiac dysfunctions caused by histamine release.