Showing papers on "In vivo published in 1973"

Acetaminophen-induced hepatic necrosis. 3. Cytochrome P-450-mediated covalent binding in vitro.

Abstract: The binding of 3 H-acetaminophen to hepatic microsomes was studied in vitro . Binding of 3 H-acetaminophen to rat and mouse microsomal protein was linear with time and with protein concentration. Binding occurred by covalent linkage to amino acids of protein. Reduced nicotinamide adenine dinueleotide phosphate and oxygen were necessary for the binding while carbon monoxide or cobaltous chloride pretreatment inhibited it, demonstrating that a cytochrome P-450-dependent. mixed function oxidase mediated the binding. The extent of in vitro binding correlated with treatments that alter hepatic necrosis and in vivo binding, indicating that in vitro binding was a valid index of acetaminophen-induced hepatotoxicity. Analogous studies with 2-acetylaminofluorene showed that its binding to microsomal protein also was dependent on cytochrome P-450. Since the toxicity of 2-acetylaminofluorene results from its conversion to an N-hydroxy derivative, the collective data are consistent with the hypothesis that the hepatotoxic metabolite of acetaminmophen may be an N-hydroxy derivative.

Platelet—tumor-cell interactions in mice. The role of platelets in the spread of malignant disease

Abstract: Thrombocytopenia reduces the number of metastases produced by a wide variety of murine tumors. Studies aimed at investigating interactions between tumors and platelets reveal that many tumors aggregated platelets in vitro and/or produced thrombocytopenia in vivo. In some instances, tumor-cell-induced thrombocytopenia in vivo was accompanied by accumulation of platelets in the lung. Thrombocytopenia was most active against metastases produced by tumors with the capacity to aggregate platelets in vitro and/or in vivo but it was also effective against metastases produced by tumors lacking such a capacity. Further studies, aimed at increasing platelet aggregation in vivo, as when fibroblasts were added to the tumor inoculum, or decreasing the platelet response to aggregating agents, as when aspirin was administered to mice, strongly support the role of platelet aggregation and the platelet release reaction in metastasis. As expected, fibroblasts enhanced while aspirin decreased tumor spread, the latter being equally effective against both artificially induced and spontaneously-occurring metastases. Formation of platelet aggregates not only enhances but also seems to change the distribution of metastases. Tumors with platelet aggregating capacities usually give lung metastases while those devoid of such a capacity may show metastases of widespread distribution. Research with 125IUDR-labelled B16 melanoma cells indicates that thrombocytopenia does not affect the initial vascular arrest of tumor cells but seems to influence their subsequent retention by the lung.

Iron-Binding Catechols and Virulence in Escherichia coli.

Abstract: Previous work suggested that virulent bacteria, which can grow rapidly in serum, must possess a specific mechanism for removing iron from its transferrin complex. Two strains of Escherichia coli were examined with this in mind. Strain O141, which showed inoculum-dependent growth in serum and multiplied in the mouse peritoneum, secreted iron-binding catechols into both synthetic medium and serum. One of these compounds has an association constant for iron similar to that of transferrin. Both transferrin and ethylenediamine-di-o-hydroxyphenyl acetic acid (EDDA), which have very high affinities for ferric iron, induced catechol synthesis in growing cultures of strain O111. This organism was inhibited by normal horse serum. Further work showed that traces of specific antibody inhibited catechol synthesis by O111 exposed to EDDA; therefore, the existence of this inhibitory process means that the organism can no longer obtain Fe3+, which all remains bound to transferrin in serum. In vivo, the inhibition of O111 is similar to that produced by serum in vitro. Neither phagocytosis nor killing by complement appeared to be of any significance during the first 4 h of the infections. Significantly, the purified catechol was capable of abolishing bacteriostasis in vivo. Since these results show that the production of iron-binding catechols is essential for rapid bacterial growth both in vitro and in vivo, these compounds should therefore be considered as true virulence factors. Conversely, any interference by the host with the production or activity of these compounds would constitute an important aspect of antibacterial defense. Images

PARENCHYMAL CELLS FROM ADULT RAT LIVER IN NONPROLIFERATING MONOLAYER CULTURE : I. Functional Studies

Abstract: Parenchymal cells from adult rat liver have been established in primary monolayer culture. Donor animals are subjected to a partial hepatectomy and, 4 days later, cells are prepared by collagenase perfusion of the regenerated liver. The hepatic parenchymal cells, separated from nonparenchymal material and suspended in serum-free medium, are placed in plastic tissue culture dishes, where they form a monolayer within 24 h. The monolayer cells exhibit minimal mitotic activity and demonstrate several major metabolic functions characteristic of liver in vivo; these include albumin synthesis and secretion, gluconeogenesis from 3-carbon precursors, responsiveness to insulin and glucagon, glycogen synthesis, and activity of two microsomal enzymes. These functions are present in the monolayer cells for several days at activities similar to those observed in the liver in vivo. The findings indicate that hepatic parenchymal cells in this monolayer system are viable and behave in many respects like normal adult rat liver.

Freeze-blowing: a new technique for the study of brain in vivo.

Abstract: — A new apparatus is described which removes and freezes brains of conscious rats more rapidly than was heretofore possible. The apparatus consists of two probes which are driven simultaneously into the cranial vault of the rat immobilized in a specially constructed restraining cage. When in position, air under pressure enters through one probe and blows the supratentorial portion of the brain tissue (situated between the olfactory bulbs and the superior colliculi) out the other probe and into a thin chamber previously cooled in liquid N2. This method stops brain tissue metabolism more rapidly than the previously-described methods of microwave irradiation, decapitation into liquid N2, or whole-animal immersion into liquid N2, as evidenced by the measurement of labile metabolites and redox states. Thus, samples of freeze-blown brain had higher levels of a-oxoglutarate, creatine phosphate, pyruvate, glucose and glucose-6-phosphate and lower levels of lactate, malate and AMP than brain tissue obtained by the other methods. The free cytoplasmic [NAD+]/[NADH2], [NADP+]/[NADPH2] and [ATP]/[ADP] [HPO42-] ratios were higher in freeze-blown samples. These data indicate that more extensive anoxic metabolism occurred when methods other than freeze-blowing were used. We conclude that the levels of metabolites measured in brain obtained with the freeze-blowing technique more closely resemble those which occur in vivo.

Trimethoprim-Sulfamethoxazole: In Vitro Microbiological Aspects

Abstract: The ultimate effect on bacteria of trimethoprim (TMP) and sulfamethoxazole (SMZ) is to deprive them of folate coenzymes; the spectra of in vitro activity of TMP and SMZ are therefore similar although TMP is usually 20-100 times more active than is SMZ. The synergy of TMP and SMZ can be demonstrated in vitro by increases in bacteriostatic and bactericidal activities. There is an optimal ratio for producing these effects, and it corresponds to the ratios of the MIC of each drug when acting alone; however, synergy does occur over a wide range of ratios. The in vitro activity of TMP-SMZ depends on the medium in which it is measured; traces of thymidine will almost completely abolish activity. The size of the inoculum also affects activity. The activity is not affected in vitro or in vivo by the presence of exogenous folates except in the case of Streptococcus faecalis. Resistance to one of the drugs as measured by conventional tests may not abolish synergy; synergy is maximal, however, when the organism is susceptible to both drugs. Multiple studies have confirmed that TMP-SMZ has a wide spectrum of in vitro activity. The only outstanding exception is Pseudomonas aeruginosa. From heavy inocula, resistance to TMP develops rapidly, but the rate is markedly reduced by the presence of SMZ. The retarding effects of the sulfonamide depend on the degree of susceptibility of the organism and become small with highly resistant strains. R-factors conferring resistance to TMP have been identified, although at present they are rare. Susceptibility testing presents no problems provided a suitable medium and a small inoculum are used. A single combined disk containing 1.25 tg of TMP and 23.75 9g of SMZ is adequate for routine tests.

A method for measuring DNA damage and repair in the liver in vivo.

Abstract: Summary Alkaline sucrose gradients were used to study the induction of single-strand breaks in and repair of rat liver DNA in vivo. Liver DNA was labeled during regeneration after partial hepatectomy. DNA was isolated from the organ in such a way as to minimize the induction of breaks due to handling or enzymatic activity. Different methods of preparing high-molecular-weight liver DNA were compared. Best results were obtained by squashing the liver in an ethylenediaminetetraacetic acid-sodium chloride buffer. Using this squash technique and alkaline sucrose gradients, high-molecular-weight DNA was regularly obtained. This DNA behaved like single-stranded DNA on hydroxyapatite columns and contained no detectable contamination of protein or RNA. It was demonstrated by the method described that hepatic DNA damage was produced within four hr by the administration of methyl methanesulfonate and that the rat can repair such hepatic DNA damage in vivo within 48 hr.

Patterns of Damage and Repair of Liver DNA Induced by Carcinogenic Methylating Agents in Vivo

Abstract: Single-strand breaks induced by methylating agents in liver DNA and their rejoining were studied in vivo by the use of alkaline sucrose gradients. DNA breaks were induced by the i.p. injection of single doses of dimethylnitrosamine (DMN), methylazoxymethanol acetate (MAM), N -methyl- N -nitrosourea (MNU), and N -methyl- N -nitrosourethan. The degree of damage was dependent on the dose of the substance given. DMN and MAM are comparably efficient in inducing marked single-strand breaks of liver DNA in small doses. MNU, although noncarcinogenic for liver, causes single-strand breaks of liver DNA, while N -methyl- N -nitrosourethan causes less damage even at toxic doses. The repair of the damage to DNA induced by MNU was complete in the first week while that induced by DMN and MAM was still not complete within 14 days after administration of the compond. Thus, the repair of the damage induced by hepatocarcinogens (DMN and MAM) seemed to be slower than that with the methylating agents not carcinogenic for the liver (MNU and methyl methanesulfonate).

Evidence that Lithium Induces Human Granulocyte Proliferation: Elevated Serum Vitamin B12 Binding Capacity in Vivo and Granulocyte Colony Proliferation in Vitro

Abstract: Summary. Concentrations of lithium similar to those present in the blood of patients receiving lithium carbonate therapy for manic-depressive psychosis cause leucocytosis in vivo and proliferation of human granulocyte colonies in vitro. These findings, plus the finding of elevated unsaturated vitamin B12 binding capacity in patients with granulocytosis induced by lithium carbonate, suggests that such therapy produces a true increase in the body granulocyte pool. The possible value of lithium therapy in neutropenic states in man requires exploration.

Beta Cell Replication in Rat Pancreatic Monolayer Cultures: Effects of Glucose, Tolbutamide, Glucocorticoid, Growth Hormone and Glucagon

Abstract: Rat pancreatic monolayer cultures were utilized to study the effects of glucose, tolbutamide, dexamethasone phosphate, bovine growth hormone and porcine glucagon on beta cell replication. Cultures were established in medium 199 (glucose concentration 1 mg. per milliliter) containing 10 per cent fetal bovine serum. They were subsequently incubated for two consecutive two day intervals in this same control medium or in media containing the test agents. Cultures were then labeled with 3-H-thymidine and reincubated in control medium for two days to permit beta cell regranulation, thus facilitating staining. Replication was estimated by determining the frequency of labeling of aldehydethionin positive (AT+) cells in stained radioautographs. Both glucose (3 mg. per milliliter medium) and tolbutamide (100 μ. per milliliter) caused a three- to fourfoldincrease in labeling compared to controls (14 labeled AT+ cells per 500 AT+ cells). In -contrast, dexamethasone (10 μ. per milliliter) produced a two-thirds decrease in labeling. Growth hormone (10 μ. per milliliter) and glucagon (10 μ per milliliter) produced no statistically significant effects. Alterations in labeling correlated with changes in the quantity of insulin released into the medium during each of the two day incubation intervals. Both glucose and tolbutamide increased insulin release; dexamethasone decreased insulin release, while growth hormone and glucagon produced no significant effects. These data suggest that agents which stimulate insulin release can trigger beta cell replication in vitro. In addition, since growth hormone, glucocorticoid and glucagon stimulate beta cell replication in vivo but not in vitro, this action in vivo may be mediated by effects on other tissues rather than by direct action on the beta cell.

Radiosensitization of anoxic cells by metronidazole

Abstract: Several drugs of high electron affinity have been identified which sensitize cells in vitro (Adams and Cooke, 1969; Adams et al., 1971; Reuvers, Chapman and Borsa, 1972). However, their in vivo use has been limited (Hornsey, Hedges and Bryant, 1968; Denekamp and Michael, 1972) and the interpretation of the results complicated by a lack of relevant toxicological and pharmacological data. It is important to know that the drug concentration reaching the hypoxic cells under study approximates that required for sensitization of mammalian cells in vitro.

In vivo and in vitro immunological cross-reactions between basic encephalitogen and synthetic basic polypeptides capable of suppressing experimental allergic encephalomyelitis.

Abstract: A significant extent of immunological cross-reactivity has been demonstrated between the basic encephalitogenic protein of bovine origin and several synthetic amino acid copolymers which have suppressive effect on experimental allergic encephalomyelitis (EAE). This cross-reactivity has been conclusively established on the cellular level, both in vivo by means of delayed hypersensitivity skin tests and in vitro using transformation of sensitized lymphocytes, as measured by incorporation of radioactive thymidine. The in vitro experiments have been conducted with lymph node cells from guinea pigs of both random bred and inbred strains, and on spleen and lymph node cells from rabbits. Definite cross-reactivity was observed between the basic encephalitogen and all the synthetic copolymers which were previously shown effective in suppression of EAE, whereas ineffective copolymers or unrelated proteins did not show any cross-reactivity. In the case of strain 2 guinea pigs and rabbit lymph node cells the cross-reactivity in vitro was manifested by direct cross-stimulation of the lymphocytes, whereas in random-bred or strain 13 guinea pigs and rabbit spleen cells, the cross-reaction was detected only by means of specific inhibition of the homologous stimulation by the heterologous antigen. A limited extent of cross-reactivity was observed on the humoral level as well; antibodies provoked in guinea pigs against the synthetic copolymer Cop 1 cross-reacted in the passive cutaneous anaphylaxis assay with the bovine basic encephalitogen.

The influx of amino acids into the brain of the rat in vivo: the essential compared with some non-essential amino acids

Abstract: The cerebral influx rates of fifteen amino acids were measured directly in living rats by means of a new technique which makes it possible to maintain a constant specific activity of a radioactively labelled amino acid in the bloodstream. A wide variation in the influx rates of the amino acids was found. These rates differed from those found by other workers using in vitro preparations, but are consistent with the theory that amino acids enter the brain mainly by carrier mediated transport processes with a high degree of specificity. There are a number of important differences between the behaviour of the transport processes in vivo and in vitro. The influx rates of the various amino acids were directly proportional to their concentrations in blood plasma (over the range of concentrations studied). All the nutritionally essential amino acids had relatively high influx rates as did other amino acids which the brain does not seem to be able to synthesize. On the other hand, amino acids that the brain can readily synthesize and two amino acids which are not normally found in mammalian tissues had low influx rates.

Comparative study of estrogen action.

Abstract: By comparing 16 estradiol derivatives, it has been possible to assess the relative influence of distribution, metabolism, uterine uptake, and plasma and tissue binding on uterotrophic activity in the rat. This comparison has revealed that all active compounds are bound in vivo to the 8 S uterus cytosol receptor, but that their activity cannot be related to binding in vitro, owing to the many factors acting on the compound in vivo prior to the triggering of the biological response. It is suggested that only the free molecule in the plasma is active on the uterus and that consequently activity is modulated by plasma binding.

Decline in Phytohemagglutinin Responsiveness of Spleen Cells from Aging Mice

Abstract: Discussion and SummaryThe present data show a dramatic age-related decrease in cell-mediated immunity as measured by in vitro3H-dThd incorporation by PHA-stimulated mouse spleen cells. Thus, not only is there a marked decline in humoral immune responsiveness during senescence (5), but it now appears that a significant decrease in cell-mediated immunity also occurs. It has been recently reported from this laboratory by Peterson et al. (18), that spleen cells from aged animals are less effective in producing acute mortality in the classical graft-versus-host reaction. Concurrently with these studies, Goodman and Makinodan (19) have observed a marked decrease in cell-mediated immune responsiveness assessed by in vivo allogeneic tumor resistance and in vitro cytolytic activity. Walford and associates (20) have also recently reported a decline in cell-mediated immunity as measured by the in vitro mixed lymphocyte reaction. The present and aforementioned systematic studies were all carried out in inbred mice, w...

Monocyte kinetic studies in normal and disease states.

Abstract: Summary. Monocyte kinetics were studied in eight normal subjects and 27 patients suffering from various diseases: acute, subacute and chronic infections, malignant tumours, Boeck's sarcoid, neutropenia, and diseases associated with chronic exanthemata or splenomegaly. Autotransfusion of blood cells labelled with 3H-diisopropylfluorophosphate in vitro was carried out and the fate of these cells in vivo was followed by autoradiographs of leucocyte concentrates prepared from venous samples. Within a few minutes the transfused monocytes equilibrated with the total blood monocyte pool (TBMP). As TBMP proved to be larger than the circulating monocyte pool (CMP), the existence of a marginal monocyte pool (MMP) was postulated. In normal subjects the CMP:MMP ratio averaged 1:3.5. Moderate deviations occurred in disease states. Monocytes left the vascular system at an exponential rate, the mean normal T4 being 8.4 hr. Slight prolongation of T4 was observed in some patients with monocytosis, the maximal values reaching 15 hr. Shortened T4 was found in one patient with acute infection (4.0 hr) and in another with gross splenomegaly (3.5 hr). The monocyte turnover rate (MTR) in normal subjects averaged 6 × 108 monocytes/hr or 7 × 106 monocytes/hr/kg. Highly significant positive correlations were evaluated between the blood monocyte count and TBMP or MTR respectively.

Protamine--antagonist to heparin.

Abstract: Protamine is used for titration of heparin in vitro for diagnosis of hemorrhagic states and for neutralization of heparin in vivo to terminate heparinization. The protamine equivalent varies with the heparin preparation, conditions of testing and, in vivo, with the amount of heparin present in the circulation. The latter depends on time after administration and the hemodynamic and metabolic state of the patient. Protamine, when injected rapidly, will release histamine and agglutinate platelets. Bleeding (spontaneous hemorrhage) demonstrates a multiple breakdown of hemostatic mechanisms due to surgical stress, drugs, exposure of the blood to foreign surfaces, etc. Simple rules for the amount of protamine required for an individual patient based on clinical judgement will be satisfactory in most cases. When hemostasis is not achieved, it must be appreciated that heparin and protamine are only part of a complex deteriorating situation.

Cyclic Adenosine Monophosphate, Metabolites, and Phosphorylase in Neural Tissue: A Comparison of Methods of Fixation

Abstract: Fixation of rat brain tissue by freeze-blowing, microwave irradiation, immersion of whole rats in liquid nitrogen, and decapitation into liquid nitrogen indicates that postmortem changes in metabolites and enzyme forms are minimal in freeze-blown brains. Cyclic adenosine monophosphate levels are lowest in microwave-irradiated brains, which has been interpreted by some investigators to indicate rapid fixation and minimal anoxia. However, the changes in phosphocreatine, adenosine triphosphate, lactate, and phosphorylase clearly demonstrate that fixation by freeze-blowing or immersion in liquid nitrogen more closely approximate the state in vivo.

Inhibition of lymphocyte cytotoxicity for human colon carcinoma by treatment with solubilized tumour membrane fractions.

Abstract: The in vitro cytotoxic action of patients' lymphocytes against colon carcinoma cells was evaluated following incubation of the lymphocytes with papain-solubilized tumour-membrane preparations. Soluble extracts of pooled colon carcinomas inhibited cytotoxicity by sensitized lymphocytes, but similar extracts of normal colon or melanoma had no inhibitory effect. The results suggest that soluble tumour antigen may play a role in abolishing lymphocyte reactivity, and this is interpreted as supporting the concept that cellular immunity against tumours in vivo may be inhibited by circulating antigen.

Bleomycin as a Possible Synchronizing Agent for Human Tumor Cells in Vivo

Abstract: At low doses bleomycin (BLM) reversibly inhibits cell progression at the S-G2 boundary Cells located in other stages of the cell cycle are essentially unaffected; therefore, when present for a complete cell cycle, BLM becomes a prospective in vivo cell-synchronizing agent In the five trials reported here, BLM was used to synchronize human malignant melanoma cells in vivo Tumor biopsies were pulse-labeled with tritiated thymidine and assayed by liquid scintillation and autoradiographic techniques Following the synchrony block, cells at the S-G2 boundary progressed to S phase in a partially synchronized wave The labeling indices indicated about 15 to 4 times the normal number of cells in S phase at the peak times following the first BLM treatment Therefore, BLM partially synchronizes cells in vivo , and this technique provides a rapid and accurate means of locating the synchronized cells and for scheduling of a second drug for a maximum effect on tumor cell killing

Bioassay and Relative Cytotoxic Potency of Cyclophosphamide Metabolites Generated in Vitro and in Vivo

Abstract: Summary Cytotoxic potencies of cyclophosphamide, 4-ketocyclophosphamide, carboxyphosphamide, mechlorethamine (HN2), bis(2-chloroethyl)amine (nor-HN2), chlorambucil, acrolein, and cyclophosphamide metabolites generated in vivo and in vitro were determined via bioassay. Our bioassay procedure was to incubate the potential cytotoxic agent with Walker 256 ascites cells in vitro , inject these cells into host rats, record survival times, estimate the number of viable cells which must have been injected to account for the observed survival time, and calculate percentage cell kill from these estimates. A log-linear relationship between tumor cell kill and exposure time or drug concentration was observed. Cyclophosphamide and 4-ketocyclophosphamide were noncytotoxic, and incubation of the latter with a microsomal or 105,000 × g supernatant fraction did not activate it. Acrolein and carboxyphosphamide were only minimally cytotoxic. HN2, chlorambucil, and nor-HN2 all were cytotoxic; HN2 was most and nor-HN2 was least potent. The cytotoxic potency of cyclophosphamide metabolites generated in vitro and in vivo was expressed as the concentration of metabolite(s) in nor-HN2 or formaldehyde equivalents that was required to kill 90% of the tumor cells. For total cyclophosphamide metabolites obtained after the incubation of cyclophosphamide with hepatic microsomes or with a 9000 × g supernatant fraction and from blood or urine after cyclophosphamide injection, the concentrations of drug required to kill 90% of the tumor cells were 0.087, 0.42, 0.66, and 4.5 µm, respectively, when expressed in nor-HN2 equivalents, and were 0.035, 0.037, 0.041, and 0.17 µm, respectively, when expressed in formaldehyde equivalents. Cyclophosphamide, 4-ketocyclophosphamide, carboxyphosphamide, nor-HN2, and acrolein could not account for the cytotoxic activity of the cyclophosphamide metabolite generated in vitro by hepatic microsomal mixedfunction oxidase action, since the latter was a more potent cytotoxic agent than any of these compounds, although it was less potent than HN2. Trapping of the microsome-generated metabolite with semicarbazide reduced its cytotoxic potency. These data support the contention that cyclophosphamide is activated (to a cytotoxic metabolite) primarily by mixed-function oxidase action of the hepatic endoplasmic reticulum which oxidizes it to aldophosphamide, and that aldophosphamide is inactivated (as a cytotoxic agent) by aldehyde oxidase (EC 1.2.3.1) and/or aldehyde dehydrogenase (EC 1.2.1.3), which oxidize(s) aldophosphamide to carboxyphosphamide. In addition, the data provide estimates of the potency of aldophosphamide as a cytotoxic agent relative to that of other alkylating agents.

In Vitro Myelosuppression and Immunosuppression by Ethanol

Abstract: A B S T R A C T Concentrations of ethanol similar to those in the blood of intoxicated patients suppressed phytohemagglutinin- or streptolysin 0-induced lymphocyte transformation, and inhibited bone marrow granulocyte colony growth in soft agar. Inhibition of lymphocyte transformation and granulocyte colony growth occurred despite the presence of large concentrations of folate and other vitamins. These in vitro findings may relate to in vivo effects of ethanol on myeloid and lymphoid tissue.