Debio 0507 primarily forms diaminocyclohexane-Pt-d(GpG) and -d(ApG) DNA adducts in HCT116 cells.

PURPOSE: To characterize the cellular action mechanism of Debio 0507, we compared the major DNA adducts formed by Debio 0507- and oxaliplatin-treated HCT116 human colon carcinoma cells by a combination of inductively coupled plasma mass spectrometry (ICP-MS) and ultraperformance liquid chromatography mass spectrometry (UPLC-MS/MS). METHODS: HCT116 cells were treated with IC(50) doses of Debio 0507 or oxaliplatin for 3 days. Total cellular Pt-DNA adducts were determined by ICP-MS. The DNA was digested, and the major Pt-DNA adducts formed by both drugs were characterized by UPLC/MS/MS essentially as described previously for cisplatin (Baskerville-Abraham et al. in Chem Res Toxicol 22:905-912, 2009). RESULTS: The Pt level/deoxynucleotide was 7.4/10(4) for DNA from Debio 0507-treated cells and 5.5/10(4) for oxaliplatin-treated cells following a 3-day treatment at the IC(50) for each drug. UPLC-MS/MS in the positive ion mode confirmed the major Pt-DNA adducts formed by both drugs were dach-Pt-d(GpG) (904.2 m/z â†’ 610 m/z and 904.2 m/z â†’ 459 m/z) and dach-Pt-d(ApG) (888.2 m/z â†’ 594 m/z and 888.2 m/z â†’ 459 m/z). CONCLUSIONS: These data show that the major DNA adducts formed by Debio 0507 are the dach-Pt-d(GpG) and dach-Pt-d(ApG) adducts and at equitoxic doses Debio 0507 and oxaliplatin form similar levels of dach-Pt-d(GpG) and dach-Pt-d(ApG) adducts. This suggests that the action mechanisms of Debio 0507 and oxaliplatin are similar at a cellular level.

Effect of human basic fibroblast growth factor on fibroblast proliferation, cell volume, collagen lattice contraction: in comparison with acidic type.

Human recombinant basic fibroblast growth factor (bFGF) is a potent mitogen for normal human dermal fibroblasts in the presence of heparin which binds to and stabilizes it. The optimal mitotic response is obtained with a concentration of 1 ng/ml of bFGF in monolayer cultures (non-differentiated fibroblasts) as well as in the better-differentiated fibroblasts obtained through the 'collagen lattices' culture system (fibroblasts embedded in a 3-dimensional collagen gel) achieving a doubling of the cell number in 8 days. Despite increasing the number of cells, bFGF decreases the ability of fibroblasts to contract collagen fibers. This inhibition is concentration-dependent and reaches a plateau at a dose of about 1 ng/ml. This effect is associated with a bFGF-induced decrease of fibroblast volume. Various dosing regimens indicate that although the highest response was obtained by daily dosing nearly optimal response was obtained either by early daily dosing or short intermittent treatment. Interestingly, the fibroblast mitotic response to bFGF decreases steadily when fibroblasts mature in collagen gels. The mitogenic properties of bFGF associated to its ability to inhibit fibroblasts contraction, if demonstrated in vivo, may be of interest in the management of wound healing.

In-vitro versus in-vivo activities of new 5-lipoxygenase inhibitors with antiinflammatory activity.

The possible relationship between in-vitro inhibition of lipoxygenase (LO)/cyclooxygenase (CO) and in-vivo antiinflammatory effects of compounds such as isoflavanes (Zy 16369, Zy 16372, Zy 16681) was investigated. The latter were all shown to be potent 5-LO inhibitors when tested in vitro on human peritoneal macrophages (IC50 = 1-7 mumol/l). Zy 16372 and Zy 16681 also inhibited the 12- and 15-LO and, to a minor extent, the CO. In order to evaluate the antiinflammatory and antiproliferative effects of these compounds in vivo they were applied topically to mice. No definite correlation could be made between the inhibition of the ear oedema induced by arachidonic acid (AA), the inhibition of the epidermal ornithine-decarboxylase (ODC) activity induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and the in-vitro activities of the compounds. Zy 16372 appeared to inhibit the oedema dose-dependently (ED50 = 5 mumol/ear) and seemed to be the most potent among the 3 compounds tested and slightly more potent than the reference compound nordihydroguaiaretic acid. As inhibitors of TPA-induced ODC, all 3 compounds exhibited comparable activity. These results suggest that the in-vivo effects of the compounds might be mediated by components other than AA metabolites, and/or be related to their specific kinetic patterns.

Postimplantation embryo culture for the assessment of the teratogenic potential and potency of compounds.

Whole rat embryos cultured during the early stages of organogenesis were subjected to a panel of selected chemicals. Of seventeen known in vivo teratogens, seventeen also induced specific malformations in embryos grown in culture. Of ten chemicals which were reported to be negative in in vivo rat teratogenicity studies, eight also did not provoke dysmorphogenic effects in vitro. Of five additionally tested retinoids, all induced multiple malformations. However, concentrations used to induce these effects varied considerably, isotretinoin inducing malformations at 10(-5) M and arotinoid at 10(-11) M. The results indicate qualitatively as well as quantitatively a high predictability of this in vitro system and suggest that the postimplantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.

Post-implantation embryo culture: validation with selected compounds for teratogenicity testing.

1. Some chemical compounds selected by experts for the validation of in vitro teratogenicity testing were investigated in whole rat embryos cultured during the early stages of organogenesis. All sixteen known in vivo teratogens tested also induced specific malformations in embryos grown in culture. 2. Of the nine compounds which were negative in in vivo rat teratogenicity studies, none provoked dysmorphogenic effects in cultured embryos. Abnormal development of the embryos was only observed with these compounds at concentrations also high enough to affect significantly overall growth and/or differentiation. 3. The results showed a high predictability of this system for the compounds tested and suggest that the post-implantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.

Application of the post-implantation rat embryo culture system to in vitro teratogenicity testing.

Originally developed for studying basic mechanisms in developmental biology, the post-implantation embryo culture system has been extended and applied to the testing of chemicals in the field of prenatal toxicology. The endpoints used in a procedure that has been developed for the routine short-term assessment of teratogenicity in vitro are growth (crown-rump length), differentiation (somite number) and morphology (together with measurement of yolk-sac growth and vascularization) in rat embryos explanted on day 9.5 of gestation and cultured for 48 hr in 100% homologous rat serum containing the test compound. Studies involving exposure to trichlorophenoxyacetic acid and to acetylsalicylic acid demonstrate the ability of this system to distinguish between non-specific toxicity and specific effects on development.

In vitro teratogenicity of acetylsalicylic acid on rat embryos: studies with various culture conditions.

Rat embryos taken at day 9.5 of gestation were exposed in vitro to acetylsalicylic acid (aspirin) using various culture conditions. It was observed that embryos were sensitive to aspirin emulsified in olive oil at concentrations greater than or equal to 150 micrograms/ml. Between 43% and 66% of the embryos exhibited multiple malformations depending on the culture medium, 100% homologous rat serum or Waymouth medium supplemented with 50% rat serum, respectively. At concentrations greater than or equal to 400 micrograms/ml aspirin induced further toxic effects on embryo growth and differentiation. When gelatin was used as the drug-delivery system, aspirin at concentrations of greater than or equal to 150 micrograms/ml induced some malformations (mainly irregular somite shapes) in 57% of the embryos cultured in Waymouth medium, but in only 13% of the embryos grown in 100% serum. At concentrations which were greater than or equal to 400 micrograms/ml aspirin induced dysmorphogenic effects in all embryos, without any concomittant toxicity.

Immunohistochemical localization of mouse testis-specific phosphoglycerate kinase (PGK-2) by monoclonal antibodies.

Monoclonal antibodies against mouse testis-specific phosphoglycerate kinase (PGK-2) were produced in order to determine immunohistochemically the onset of PGK-2 synthesis in the germinal epithelium of the mouse. PGK-2 was detected in testis sections in spermatids as early as stage 12 and in spermatozoa, but not in earlier stages of spermatogenesis nor in any somatic cells of the testis. During ontogeny, PGK-2 appears within the testis at day 30 post-partum, concomitant with spermatids entering the maturation phase. All three allelic isozymes PGK-2A, -2B, and -2C were detected equally by the monoclonal antibody in testis sections of several inbred mouse strains, each of which expresses a specific PGK-2 variant. Moreover, the monoclonal antibody against mouse PGK-2 reacted with heterologous sperm-specific PGK from rat, rabbit, and bull and, therefore, may serve as a useful immunochemical marker for mammalian spermatogenesis.

T cell recognition of Moloney sarcoma virus proteins. II. Phenotypes of the different lymphocyte subpopulations producing and responding to blastogenic factors and their relative frequencies during tumor regression.

The characteristics of the blastogenic response of splenic lymphocytes from MoLV/MSV immune mice was examined. Splenic lymphocytes from immune mice proliferate in vitro in response to the major virion envelope glycoprotein, gp70. Associated with this response is production of blastogenic factors that induce the proliferation of nylon wool-purified lymphocytes from either normal or immune mice. The phenotype of antigen-specific lymphocytes essential for the production of blastogenic factors was found to be Thy 1.2+, Lyt 1+, 2-. The induction of proliferation by blastogenic factors does not require the presence of antigen. Using splenic lymphocytes from either normal or immune mice, blastogenic factors predominantly induce the proliferation of a Thy 1.2-, Lyt 1-, 2- lymphocyte. As a consequence of responding to blastogenic factors, in vitro, approximately 50% of this lymphocyte population becomes Thy 1.2+, Lyt 1+ over a period of 3 days. The frequencies of lymphocytes producing and responding to blastogenic factors were found to show characteristic changes during the course of tumor growth and regression.

Chromosomal location of the genes for human immunoglobulin heavy chains.

We have studied somatic cell hybrids between P3x63Ag8 mouse myeloma cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and either human peripheral lymphocytes or human lymphoblastoid or myeloma cells for the production of human immunoglobulin chains and for the expression of enzyme markers assigned to each of the different human chromosomes. Human chromosome 14 was the only human chromosome present in all independent hybrids producing mu, gamma, and alpha human heavy chains. In two of the independent hybrids that produced human heavy chains, human chromosome 14 was the only human chromosome present in the hybrid cells. Loss of human chromosome 14 from these hybrids resulted in the concomitant loss of their ability to produce human immunoglobulin heavy chains. In view of these results, we conclude that the genes for human immunoglobulin heavy chains are located on human chromosome 14 in immunoglobulin-producing human cells.

Antibody response to simian virus 40 tumor antigen in nude mice reconstituted with T cells.

Athymic BALB/c nude mice (nu/nu) fail to generate circulating antibodies to simian virus 40 (SV40) tumor (T) antigen when immunized with SV40-transformed mouse cells or with T antigen positive somatic cell hybrids derived from SV40-transformed human and normal mouse parental cells. However, normal BALB/c mice readily produce antibodies to SV40 T antigen. When nude mice were reconstituted with normal syngeneic T lymphocytes from spleen or thymus source, the humoral immune responsiveness to SV40 T antigen was restored.

Somatic cell hybrids between mouse peritoneal macrophages and SV40-transformed human cells. III. Identification of surface antigens coded for by human chromosomes 7 and 17.

Somatic cell hybrids between SV40-transformed human cell lines and mouse peritoneal macrophages (MPM) containing either human chromosome 7 or 17 carrying the SV40 genome were injected into mice syngeneic to the mouse parental cells. Since either chromosome 7 or 17 was the only human chromosome present in the hybrids used as immunogens, the humoral immune response to gene products coded for by either chromosome was assayed. Using a sensitive radioimmunoassay, we were able to identify noncross-reactive cell-surface antigen(s) specifically coded for by either human chromosome 7 or 17, and present in normal, tumor-derived and virus-transformed human cells. However, no reactivity against SV40 tumor-specific surface antigen (TSSA) could be detected in the antisera.