Differential effect of growth- and differentiation-inducing factors on the release of eicosanoids and phospholipids from ML-1 human myeloblastic leukemia cells.

In the absence of serum, growth of ML-1 human myeloblastic leukemia cells is induced by the insulin-like growth factor-1 (IGF1) together with transferrin (Tf), whereas monocytic differentiation is initiated by the transforming growth factor-beta (TGF-beta) in combination with Tf. Initiation of growth was followed by the rapid release of arachidonic acid (AA), hydroxyeicosatetraenoic acids (HETEs) and phospholipids into the culture medium. In contrast, induction of differentiation occurred without the release of these lipids beyond the level present in control. Inhibitors of enzymes involved in the formation of AA and of HETEs, including phospholipase A2 and lipoxygenases, caused interference with growth but not with differentiation, and an inhibitor of the cyclooxygenase path affected neither growth nor differentiation. These results indicate that the initiation of ML-1 cell growth but not of cell differentiation is dependent upon the increased formation of AA and its derivatives formed primarily via the lipoxygenase path.

Effects of photodynamic treatment of platelets or endothelial cells in vitro on platelet aggregation.

The purpose of this work was to gain insight into the role played by platelets and endothelial cells in the development of thrombogenic vascular events, observed after in vivo photodynamic therapy (PDT), by studying the in vitro effects of PDT on isolated human platelets and cultured human and bovine endothelial cells. Exposure to Photofrin II (PII) and light caused platelets to rapidly lose their ability to aggregate. Photofrin II alone at high concentrations also exerted inhibitory effects on aggregation. Endothelial cells exposed to PII- and phthalocyanine (GaCl-PcS2,3 or Zn-PCS1,2)-mediated PDT released potent platelet anti- and disaggregating activity which could be identified as prostacyclin by the following criteria: a close correlation between the time and dose dependent anti-aggregating effects and released 6-keto-PGF1 alpha (the spontaneous hydrolysis product of PGI2, determined by radioimmunoassay), the inhibition of these effects by indomethacin, accumulation of 6-keto-PGF1 alpha metabolite in the media of cells treated with PDT (as determined by HPLC analysis), and the absence of evidence for significant nitric oxide production. This prostacyclin release occurred following plasma membrane damage. Although no pro-aggregating activity was observed, endothelial cells were found to release considerable amounts of arachidonic acid and prostaglandin F2 alpha in response to PDT. These data, which indicate powerful anti-thrombogenic effects in vitro, are in sharp contrast to the vascular effects of PDT in vivo which are characterized by severe platelet aggregation, and imply that the in vivo effects involve additional components of the vascular system.

Elevated pentose cycle and glucuronyltransferase in daunorubicin-resistant P388 cells.

Anthracycline resistance of P388 daunorubicin-resistant cells cannot be accounted for merely by differences in drug uptake and retention; protection against intracellular drug was also indicated. Cytotoxicity of daunorubicin may be partially due to the formation of free radicals and reactive oxygen species (hydrogen peroxide, hydroxyl radical, singlet oxygen, and superoxide anion radical). Protection against free radicals and peroxides is largely dependent upon the availability of reduced glutathione, which in turn requires NADPH for its continual regeneration. Pentose phosphate cycle (also called hexose monophosphate shunt) is known to provide NADPH for maintenance of glutathione. Activities of the two NADPH-producing dehydrogenases of the cycle, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, were 40% higher (P less than 0.05) and activity of the cycle in intact cells was 2-fold higher in the resistant than the sensitive cells. The cycle was as active in these cells as it is known to be in macrophages, indicating a very effective protection against oxidative stress, free radicals, and alkylating electrophiles. Elevated activity of the pentose phosphate pathway in drug-resistant cells can represent a mechanism of resistance against multiple structurally unrelated drugs. Efflux of daunorubicin may be aided by further metabolism to glucuronides. Daunorubicinol, a known active metabolite of daunorubicin, can be metabolized to a glucuronide by the cells and eliminated into the surrounding medium. Glucuronidation of daunorubicinol was evidenced by (a) release of daunorubicinol following glucuronidase hydrolysis of media from cell incubations with 1.8 microM daunorubicin and (b) production of radioactive glucuronide when cell homogenates were incubated with UDP-[14C]glucuronic acid plus daunorubicinol. Glucuronyltransferase activity with a broad substrate specificity was found in the cells. Using model substrates, 1-naphthol and o-aminophenol, it was determined that glucuronyltransferase activity was 4 times higher in daunorubicin-resistant than -sensitive P388 cells. Elevated glucuronyltransferase could contribute to daunorubicin and multidrug resistance.

Coinduction of glucose-regulated proteins and doxorubicin resistance in Chinese hamster cells.

The glucose-regulated protein (GRP) system in mammalian cells is induced by glucose deprivation, anoxia, the calcium ionophore A23187, and 2-deoxyglucose. In Chinese hamster ovary cells the major GRPs are approximately equal to 76, 97, and 170 kDa. Removal of each of these four GRP-inducing stresses leads to the coordinate repression of GRPs and induction of the major heat shock proteins at 70 and 89 kDa. The application of each of these four GRP-inducing conditions leads to a significant induction of resistance to the drug doxorubicin. Removal of each GRP-inducing condition results in the rapid disappearance of this resistance in a manner that correlates with the repression of the GRPs. The retention of doxorubicin by GRP-induced cells does not explain the induced drug resistance. When the RIF in vitro/in vivo tumor system is probed with an antibody against the 76-kDa GRP, a significant increase in this GRP is observed in cells obtained from the central regions of tumors. Since hypoxia and/or nutrient deprivation can occur during tumor development, a GRP-induced state in the tumor may confer resistance to doxorubicin treatment.

Plasma levels of daunorubicin metabolites and the outcome of ANLL therapy.

Levels of plasma daunorubicin, daunorubicinol and aglycone metabolites were measured in 47 patients 3 h after daunorubicin was administered daily for three days as part of a cytosine arabinoside/daunorubicin remission induction regimen. High-pressure liquid chromatography with fluorescence detection was used for separation and quantitation of the drug and its metabolites. A wide range of plasma levels were observed regardless of the outcome of therapy. Patients who had high levels of the drug, or daunorubicinol on day 1 of therapy tended to have high levels on days 2 and 3 of the regimen. Three hours after the third daily dose of daunorubicin was administered, patients who would not enter remission had significantly higher levels of aglycone metabolites in plasma than did patients who entered remission. These data indicate that resistance to chemotherapeutic effects of daunorubicin may be connected with metabolism of the drug, especially with enhanced metabolism to aglycones.

Relationship between plasma adriamycin levels and the outcome of remission induction therapy for acute nonlymphocytic leukemia.

Plasma adriamycin and adriamycinol levels were measured in 45 patients with acute nonlymphocytic leukemia 3 h after the drug was administered. A wide range of levels as found. Plasma levels increased after the administration of each of the three daily doses of the drug. High plasma levels were associated with both death during remission induction therapy and, for patients who entered remission, long remissions.

Formation of glucuronide, sulphate and glutathione conjugates of benzo[a]pyrene metabolites in hepatocytes isolated from inbred strains of mice.

Metabolism of benzo[a]pyrene (BP) was studied in mouse hepatocytes isolated from uninduced animals of C57BL/6 Jacobs (B6) and C3Hf/HeHa (C3) inbred strains. Conjugates with sulphate, glucuronate and glutathione were the major products of BP biotransformation in the intact cells. Their formation was measured by determining the radioactivity incorporated from [3H]BP into the appropriate metabolite, after separation on silica gel t.l.c. plates. The conjugates were identified by their susceptibility to the action of specific degrading enzymes, arylsulphatase, beta-glucuronidase and gamma-glutamyltransferase. Effects of inhibitors of conjugation were also examined. D-Galactosamine and diethyl maleate caused approximately 50% inhibition of the formation of glucuronide and glutathione derivatives of BP, respectively. The effect of salicylamide was less specific, besides an 88% decrease in sulphation of BP metabolites, a 40% decrease in the formation of glutathione conjugates was observed in the presence of this inhibitor. In hepatocytes of B6 mouse, all the above three types of BP conjugates were formed in almost equimolar quantities. The total formation of BP conjugates was 42% higher in B6 hepatocytes than in those of C3 strain. The most significant difference (1.7-fold) was in the production of BP glucuronides, despite an absence of observable differences between these mouse strains in the activity of microsomal UDP-glucuronosyltransferase and in the rate of 1-naphthol conjugation in isolated hepatocytes. Simultaneously, 2.5-fold higher accumulation of unconjugated BP metabolites was observed in the hepatocyte suspension of B6 than C3 strain and a 1.4-fold higher activity of aryl hydrocarbon hydroxylase in hepatic microsomes of this strain. The unconjugated metabolites of BP were separated into four major fractions by h.p.l.c. The retention times of the metabolites corresponded to trans 9,10-diol; trans 7,8-diol; 9-hydroxy- and 3-hydroxy-BP. Despite quantitative differences between B6 and C3 strains of mice in BP metabolism, the same degree of covalent binding of BP metabolites to cellular DNA, was observed. The results indicate a relatively high capacity of hepatocytes from uninduced mice for conjugation of BP metabolites. Hepatocytes isolated from various strains of mice, should be useful in elucidating the role of numerous factors in metabolism and biologic activity of BP and related carcinogens.

A simplified method for determination of daunorubicin, adriamycin, and their chief fluorescent metabolites in human plasma by high-pressure liquid chromatography.

A simple method was developed for the routine monitoring of daunorubicin (DR) or adriamycin (ADR) and of their chief fluorescent metabolites in plasma of cancer patients. The plasma samples were treated with ethanol: hydrochloric acid mixture, following which the drug and its metabolites, released to the 40,000 g supernatant, were analyzed by HPLC. A mu-bondapak-phenyl column was used and an isocratic mobile phase consisting of acetonitrile in 0.1 M ammonium-formate buffer at pH 4.0. Average recovery of all the tested compounds within the concentration range of 17-3,450 pmol/ml plasma was 108 +/- 5% (mean +/- SD). The method was applied to analyses of plasma samples of several patients treated with DR or ADR. At 3 h after treatment with a DR dose of 45 mg/m2 or 60 mg/m2, daunorubicinol was the major metabolite and its concentrations were 46-270 or 85-305 pmol/ml, respectively; the unchanged drug was present at concentrations of 16-99 or 30-101 pmol/ml, respectively. Deoxydaunorubicinolone and deoxydaunorubicinone were detected at concentrations ranging from 0 to 89 pmoles/ml in the plasma of some patients. Plasma of patients treated with ADR (30 mg/m2) contained adriamycinol as the main detectable metabolite, but at 3 h after treatment its concentration was usually lower than that of the unchanged drug (22 +/- 9 vs 53 +/- 16 pmol/ml). Traces of 7-deoxyaglycones were detected in some plasma samples.

Absence of seasonal variation in antipyrine metabolism.

The induced activity of aryl hydrocarbon hydroxylase (AHH), measured by the metabolism of benzo[a]pyrene to fluorescent products in cultured human lymphocytes, shows a strong seasonal variation. The in vivo metabolism of antipyrine, which is also catalyzed by microsomal cytochrome P-450-dependent monooxygenases, has been reported to be correlated with AHH inducibility in human lymphocytes. To determine whether antipyrine metabolism also showed seasonal changes, we measured antipyrine half-life (t 1/2) in 10 nonsmokers and eight smokers at the two times of the year that correspond to the high and low peaks of inducible AHH activity as measured in lymphocytes. The mean antipyrine t 1/2 determined in all 18 subjects in summer was almost identical to that found in winter (mean +/- SEM = 10.90 +/- 0.65 and 10.96 +/- 0.78 hr). AHH activity in cultured human lymphocytes from the nonsmoking subjects was determined in control and 3-methylcholanthrene-induced cells to obtained inducibility ratios of 4.2 +/- 0.56 (SEM) in the summer and 1.4 +/- 0.14 (SEM) in winter. These results indicate that the seasonal variation in AHH inducibility in human lymphocytes is not reflected by a corresponding seasonal variation in antipyrine metabolism in vivo.

High-pressure liquid chromatographic analysis of benzo(a)pyrene metabolism by human lymphocytes from donors of different aryl hydrocarbon hydroxylase inducibility and antipyrine half-lives.

With high-pressure liquid chromatography (HPLC), lymphocytes from six human donors were evaluated for their ability to metabolize benzo(a)pyrene (BP). Donors whose aryl hydrocarbon hydroxylase (AHH) inducibility ratios ranged from 2.4 to 4.6 and whose antipyrine plasma half-lives ranged from 8 to 17 hr were examined. The BP metabolites identified were: 7,8-dihydrodiol, quinones, and 9-hydroxy and 3-hydroxy phenols. HPLC profiles of BP metabolites elaborated by uninduced (control) and benz(a)anthracene-induced lymphocytes were qualitatively similar among the six donors. A good correlation (r = 0.79) was found between known AHH inducibility ratios for the donors, as determined by the conventional fluorometric AHH assay, and induction of BP phenol production quantitated from HPLC data. HPLC results also indicated that the induction of benzo(a)pyrene-7,8-dihydrodiol, the proposed proximate carcinogenic form of BP, did not parallel BP phenol induction. Furthermore, the data also indicated a good negative correlation between AHH inducibility and the measurements of plasma antipyrine or urinary 4-hydroxyantipyrine half-lives (r = -0.88 or -0.91), respectively.

Gastric excretion of intravenously administered meperidine in surgical patients.

We have demonstrated sequestration of unchanged meperidine in gastric juice of humans following intravenous administration. Concentrations attained were as great as 360 times plasma concentration. The drug was first detected between 5 and 15 minutes after administration. This phenomenon appears to be due to ionization and ion trapping of meperidine in gastric juice.

Toxocara-specific antibody in the serum and aqueous humor of a patient with presumed ocular and visceral toxocariasis.

Rarely have concurrent ocular and systemic toxocariasis been reported in the literature. We describe a patient with serologically proven visceral toxocariasis who had a granulomatous lesion in the iris, small rod-like lesions in the retina, and in whom Toxocara-specific antibodies were also demonstrated in the aqueous humor. Two older siblings of this patient also had demonstrable serum antibody to Toxocara. The ocular manifestations resolved rapidly with corticosteroid and thiabendazole therapy and the initial leucocytosis, hepatomegaly, and elevated IgM level were normal at 3.5 months. These changes might be attributed to either the thiabendazole and prednisone therapy or to the natural history of this disease.

Occurrence of steroid glucuronyltransferases in a hepatoma.

Occurrence of estrone, estradiol, and testosterone glucuronyltranferase activities was tested in a well-differentiated hepatoma, Reuber H35. Transferase activities for estrone and estradiol were found in the hepatoma. The Michaelis-Menten kinetics of these two microsomal glucuronyltransferase activities were similar in hepatoma and in liver preparations, except for a somewhat higher apparent Km for estradiol in the hepatoma preparations. Under the same experimental conditions, only trace amounts of testosterone glucuronyltransferase activity could be detected in the hepatoma preparations. By contrast, in liver microsomal preparations, testosterone glucuronyltransferase activity was the highest among the steroid glucuronyltransferase activities tested.

Studies of the effects of cyclophosphamide, vincristine, and prednisone on some hepatic oxidations and conjugations.

The effects of low and high doses of three anticancer agents, cyclophosphamide, vincristine, and prednisone (given individually or in various combinations), on oxidative and conjugation pathways were studied in Sprague-Dawley male rats. Cyclophosphamide used alone at low doses decreased aniline hydroxylase and ethylmorphine demethylase activities by about 20% and at high doses produced a 30%-50% decrease in the specific activities of several microsomal mixed-function oxygenase activities, in the contents of cytochromes P-450 and b5, and in the magnitudes of type I and II drug-binding spectrum. The levels of microsomal glucouronidase, glucuronyl transferase, and sulfatase per gram of liver were also decreased (30%-50%) by the high dose of cyclophosphamide. The high dose of cyclophosphamide in conjunction with either vincristine or prednisone also produced a noticeable decrease in several activities tested; however, when cyclophosphamide was given at either low or high doses in combination with vincristine and prednisone, the activities tested were comparable to those seen in untreated controls. The mechanism of this protection is presently unknown. Vincristine, at both low and high doses, produced little effect on oxidative pathways; however, at low doses it caused a significant increase (80%) in the specific activity of hepatic microsomal sulfatase. This effect was also discernible when vincristine was given in combination with cyclophosphamide and prednisone. Other than producing a 15% decrease in liver weight and a 40% decrease in the specific activity of microsomal glucuronidase, the high dose of prednisone used had no effect on various activities tested. Results of these studies indicate a potential for drug interaction among anticancer agents and supportive drugs used in combination cancer chemotherapy.

Studies of mammalian glucoside conjugation.

The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis-Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3alpha-ol-20beta-one (3alpha-hydroxypregnan-20-beta-one)>oestradiol-17beta 3-methyl ether>oestradiol-17beta>oestriol>pregnane-3alpha,20beta-diol>oestrone. Pregnan-3alpha-ol-20beta-one, pregnane-3alpha,20beta-diol and oestrone had negligible effect on glucuronidation.