[Health foods: definitions, status, public health. Round table no. 4. XV]. / Alicaments: définitions, statuts, apports en Santé Publique. Table Ronde no 4 de Giens. XV.

'Alicaments' ('ali' for 'aliment' = 'food' in French, and 'cament' for medicament = drug), is an inappropriate term to designate a developing domain: functional foods, in other words health claims in nutrition. It is not easy to delineate the frontier between foods and drugs; this results in some regulatory problems. The key question is the evaluation of health claims. In this report, we synthesize the discussions of a workshop bringing together specialists from the food industry and nutritionists, scientists and representatives of public health departments.

New alpha-amino phosphonic acid derivatives of vinblastine: chemistry and antitumor activity.

A series of new amino phosphonic acid derivatives of vinblastine (1, VLB) has been synthesized and tested in vitro and in vivo for antitumor activity. The compounds were obtained from O4-deacetyl-VLB azide. All of the new products studied were capable of inhibiting tubulin polymerization in vitro. The most potent antitumor compounds bore an alkyl substituent on the phosphonate. In these compounds, the anti-tumor activity strongly depended on the stereochemistry of the phosphonate. The phosphonate (1S)-[1-[( O4-deacetyl-3-de(methoxycarbonyl)vincaleukoblastin-3-yl] carbonyl]amino]-2-methylpropyl]phosphonic acid diethyl ester exhibited a remarkable activity against cancer cell lines both in vitro and in vivo.

The A.H.E.A.D. method: a quantitative automated measurement of the hematotoxicity of anticancer drugs.

Among the limiting side-effects of anticancer drugs, hematological toxicity is the most frequent. Hematological analysis focuses on nucleated blood cells from mouse peripheral blood or hematopoietic organs (bone marrow, spleen, thymus). We have developed an automated method called A.H.E.A.D. (Automated Hematotoxicological Evaluation of Anticancer Drugs) allowing us routinely to screen chemical products for hematotoxicity, simultaneously with in vivo and in vitro antitumoral assays. Data are collected after a simple dilution of biological samples without any additional isolation, enrichment or purification process. The measurements are performed through an electronic Coulter counter and a slightly modified pulse-height model C1000 analyzer connected on-line to a computer. In addition to nucleated cell counts, this method allows us to get information about cell volume distribution within a given population of leukocytes. In bone marrow, a single assay provides simultaneous information on the relative contents of red, lymphoid, polymorphonuclear and blast cells. We present results obtained with this method during the hematological toxicity studies of some standard anticancer drugs. Besides drug induced alterations, the A.H.E.A.D. method allows monitoring of the recovery of hematological tissues characterization of growing populations of precursors renewing the hematopoietic organs.

Modification of membrane permeability measured by Texas-Red during cell cycle progression and differentiation.

Membrane permeability is a critical parameter which must be assessed in the course of new potential anticancer drug studies. Flow cytometry allows us to monitor the permeability status of living cells through the measurement of fluorochrome retention; conventionally this is done using fluorescein diacetate or Hoechst33342. In the present study we show that Texas-Red (TR) can be advantageously used for the same purpose because: (i) it possesses fluorescence spectra compatible with the simultaneous use of various fluorochromes; (ii) staining is rapid; (iii) it is a viability marker. TR uptake is realized through an active, energy dependent transport across the membrane. This new technique was applied to exponentially growing HL60 and their differentiated counterparts. We show that membrane permeability varies throughout the cell cycle progression and during differentiation. We also show that TR uptake is different between sensitive and cytotoxic drug resistant cells. This technique could be used, in anticancer pharmacology, to correlate phase specificity of cytotoxic drugs with membrane permeability, to understand better the mechanism of action of differentiating agents, and to document the apparition of resistant clones within populations of cells.

Pharmacological properties of a new alpha-aminophosphonic acid derivative of vinblastine.

S 12363 is a new vinca alkaloid derivative obtained by grafting an optically active alpha-aminophosphonate at the C23 position of O4-deacetyl vinblastine. This compound was as potent as Vincristine (VCR), and less potent than Vinblastine (VLB), in inhibiting in vitro tubulin polymerization. However, S 12363 was found to be 7 to 553 and 12 to 74-fold more cytotoxic than VCR and VLB, respectively, when tested on a panel of 2 murine and 6 human tumor cell lines using the Microculture Tetrazolium Assay. S 12362, which differs only by the configuration of the asymmetric carbon atom of the side chain, was 18 to 59-fold less cytotoxic. At equitoxic doses, all these compounds induced a "G2 + M" phase accumulation of L1210 cells, suggesting a similar mechanism of action. S 12363, administered i.p. or i.v., was at least as active as reference compounds on two murine transplantable tumors (P388 leukemia and B16 melanoma) while the optimal dosage was 20-fold lower: 0.15-0.20 mg/kg versus 2-5 mg/kg, respectively. S 12362 was practically inactive at 1-3 mg/kg. The hematological toxicity of S 12363 (0.1 mg/kg) was similar to that of VLB (4 mg/kg). The exceptionally high potency of S 12363 did not appear to be due to a better interaction with tubulin, its intracellular target, but rather to some properties conferred by the alpha-aminophosphonic acid, such as a facilitated uptake and/or a better cellular retention.

Use of the main tyrosine protein kinase activity purified from HL-60 in the search for a new class of anticancer compounds.

A tyrosine protein kinase (TPK) partially purified and two co-purified proteins (pp39 and pp44) from HL-60 cells were used as a basis for a SDS-PAGE electrophoresis screening test of inhibitors of the activity catalyzed by this enzyme. Such inhibitors may constitute a new class of anticancer agents. TPK is a protein of about 32 to 35 kDa as measured by gel-sizing exclusion. This enzyme is apparently the main tyrosine protein kinase from the cytosol of HL-60 cells. The test was assessed using known inhibitors of various types of protein kinases. Erbstatin and Cibcron Blue--a nucleotide analog--were shown to be potent inhibitors of TPK activity.

Effects of the new nitrosourea derivative, fotemustine, on the glutathione reductase activity in rat tissues in vivo and in isolated rat hepatocytes.

Fotemustine, a new clinically active nitrosourea, is demonstrated herein to be a poor inhibitor of glutathione reductase activity from rat liver, lung and kidney cytosols. In order to show that an intracellular step of activation does not lead to a toxic intermediary metabolite, rat hepatocytes were incubated with fotemustine. Their glutathione-related pathways were checked and shown not to be altered, while under similar experimental conditions BCNU was shown to be dramatically harmful. Furthermore, association of fotemustine with a H2O2 production leading drug, diquat, was shown to be inefficient--while BCNU is efficient--in potentiating the diquat toxicity. Considering the role of glutathione level in the detoxification of mutagens and carcinogens, the advantage of fotemustine over BCNU in therapeutic use seems substantiated.

Partial purification and characterization of a new p36/40 tyrosine protein kinase from HL-60.

A major peak of tyrosine protein kinase activity was partially purified from a Triton X100 extract of HL-60. This preparation submitted to high pressure gel filtration was eluted at a volume corresponding to a mass of 35/40 kD. This activity was insensitive to EGF and insulin. Autoradiographs of the preparations incubated with [gamma P32]-ATP and separated by electrophoresis do not give any evidence that autophosphorylation occurs for that particular tyrosine protein kinase. Furthermore, we failed to immunoprecipitate the enzyme with a specific antiphosphotyrosine antibody and anti v-src antibody. All the data presented herein suggest that this enzyme has not been previously purified.

Adult hemoglobins are synthesized in yolk sac microenvironment obtained from murine cultured blastocysts.

The capability of yolk sac (YS) hematopoietic stem cells to produce in vitro either primitive or definite erythrocytes under the influence of stimulating agents was analyzed. Two systems were used: either 9-day-old yolk sacs were explanted and cultured or 3-day-old blastocysts were cultured under conditions in which they give rise only to YS tissue. In both systems, YS tissue produced only primitive erythrocytes. When the stimulating agents (spleen cell-conditioned medium or erythropoietin) were added to the medium, YS cultures gave rise to definitive erythrocytes. These data confirm the intrinsic ability of the YS stem cells to undergo primitive erythropoiesis; furthermore, they provide evidence that YS hemopoietic progenitor cells do not require any direct interaction with cells of the embryo to undergo definitive erythropoiesis but only stimulation by agents, such as pokeweed mitogen spleen conditioned medium or erythropoietin; the YS microenvironment does not counteract this capacity.

In vitro induction of adult erythropoiesis in early mouse yolk sac.

The capacity of yolk sac hemopoietic cells to produce either primitive or definitive erythrocytes was analyzed in vitro under three different experimental conditions. (i) Before the 28-somite stage (prior to colonization of the liver rudiment by hemopoietic cells), yolk sac explanted alone produced solely primitive erythrocytes and only for a short time. (ii) When allowed to colonize a liver rudiment, hemopoietic cells from the yolk sac gave rise to definitive erythrocytes. (iii) These cells could express the same capacity when stimulated by various intraembryonic organs, even if no direct cell--cell contact was established between stimulating tissue and target hemopoietic cells. These results provide evidence that humoral factors present in embryos past the 28-somite stage act on hemopoietic cells, inducing the onset of definitive erythropoiesis.

Presence of multipotential hemopoietic cells in teratocarcinoma cultures.

Previous investigations have shown that the teratocarcinoma line PCC3/A/1 is able to differentiate in vitro to produce red blood cells of the primitive hemopoietic cell population of the mouse embryo. The present paper demonstrates the presence in the same cultures of multipotential cells capable of giving rise to colonies containing erythrocytes, macrophages, and megakaryocytes when stimulated by pokeweed-mitogen spleen-cell conditioned medium.