DNA damage in intact cells induced by bacterial metabolites of chloramphenicol.

Four chloramphenicol (CAP) metabolites known to be produced by intestinal bacteria were examined with respect to their capacity to induce DNA damage in intact cells. The induction of DNA single-strand breaks in Raji cells, activated human lymphocytes, and human marrow cells was assayed by the alkaline elution technique. One of the four compounds tested, dehydro-CAP, was capable of inducing DNA single-strand breaks in all three cell systems at concentrations of 10(-4) M. This effect is comparable to that observed previously with nitroso-CAP, the nitroreduction intermediate of CAP. The nitroreduction of dehydro-CAP by human bone marrow cell homogenate was detected by the production of the corresponding amino derivative amounting to 5.6 X 10(-5) M from 2 X 10(-3) M substrate under aerobic conditions. In sharp contrast, nitroreduction of CAP by bone marrow could not be demonstrated. The genotoxicity of dehydro-CAP, its relative stability compared to the nitroso-CAP, and its nitroreducibility by bone marrow suggest that this bacterial metabolite of CAP may play a key role as a mediator of aplastic anemia in the predisposed host.

Chloramphenicol-induced bone marrow injury: possible role of bacterial metabolites of chloramphenicol.

To explore the potential role of some bacterial metabolites of chloramphenicol (CAP) in CAP-induced hematotoxicity, we examined their cytotoxic effects on bone marrow cells in vitro using a number of cytotoxicity parameters. Among the metabolites tested, dehydro-CAP (DHCAP) and p-nitrophenyl-2-amino-3 hydroxypropanone-HCI (NPAP) were more toxic than CAP. DHCAP was at least as toxic as nitroso-CAP. At concentrations of less than or equal to 10(-4) mol/L, DHCAP caused total irreversible inhibition of myeloid colony (CFU-GM) growth and 80% inhibition of DNA synthesis in human bone marrow. Incubation of human bone marrow cells with 10(-4) mol/L nitroso-CAP or DHCAP for 24 hours resulted in 75% and 65% cell death respectively. Although DHCAP was 10- to 20-fold more cytotoxic than CAP, it was only one third as effective in inhibiting mitochondrial protein synthesis, indicating that DHCAP exerts its toxic effect by alternate mechanisms. The cytotoxicity of DHCAP and its relative stability, compared to the unstable nitroso CAP, suggest that this bacterial metabolite of CAP, and possibly others, may play a significant role in CAP-induced hematotoxicity.

DNA damage induced by chloramphenicol and its nitroso derivative: damage in intact cells.

We have postulated that the p-NO2 group of chloramphenicol (CAP) is the structural feature underlying aplastic anemia from this drug. In a series of studies to examine this hypothesis we have demonstrated the toxic nature of the CAP-reduction intermediate nitroso CAP (NO-CAP) and its damaging effect on isolated DNA in vitro. The present study was designed to examine the comparative effects of CAP, NO-CAP, and thiamphenicol (TAP) on the integrity of DNA in intact cells. By using the alkaline elution technique of Kohn, DNA damage in the form of single strand breaks could be readily demonstrated in cultured Raji cells and in PHA-stimulated normal human lymphocytes by small concentrations of NO-CAP (0.05-0.1 mM). A small but reproducible effect was observed from large concentrations of CAP (2 mM). In contrast, TAP, lacking the p-NO2 group, was without effect.

Differentiation of cultured promyelocytic leukemia cells (HL-60) induced by endotoxin-treated human lung conditioned medium.

Serum-free conditioned medium prepared from endotoxin-treated human lung tissue contains potent differentiation activity (DA) which induces the differentiation of human promyelocytic leukemia cells (HL-60) to mature functioning granulocytes and macrophages. Upon fractionation, the DA can be separated from colony-stimulating factors (CSFs). The estimated molecular weight of DA is 23,000 d which is similar to CSF-II but is much smaller than CSF-I (50,000 d). The isoelectric point (PI) value obtained from preparative isoelectrofocusing is 5.2, which is higher than CSF-I of 4.1-4.8 but lower than CSF-II of 5.6. Using isopropanol as solvent and trifluoroacetic acid (0.2%) as ion-pair, the DA is eluted at 37% isopropanol from the C-3 column in comparison to 30% for CSF-II and 34% for CSF-I.

Inhibition by rhodamine 123 of protein synthesis in mitochondria of normal and cancer tissues.

The effect of the mitochondrial dye rhodamine 123 (Rho 123) on protein synthesis (PS) activity was investigated in mitochondria isolated from liver and from both chloroma and erythroleukemia tumors. Incorporation of labelled leucine into mitochondrial protein was used to measure the rate of PS. While PS specific activity was much higher in hematopoietic tumors mitochondria as compared to that of liver, the addition of increased concentration of Rho 123 in all tested organelles resulted in increased inhibition of PS to reach 75-82% with 10 micrograms/ml of the dye. Similar results were obtained with 10 micrograms/ml of chloramphenicol, the specific inhibitor of mitochondrial PS. Moreover, under the conditions of the study, the addition of Rho 123 to mitochondria did not trigger any ATPase activity, thus eliminating any competition for the energy source ATP between PS and ATPase. These results demonstrate that, in addition to its known inhibitory action on oxidative phosphorylation, the mitochondrial dye Rho 123 has a potent inhibitory effect on PS in both liver and hematopoietic tumors mitochondria.

DNA damage induced by chloramphenicol and nitroso-chloramphenicol: protection by N-acetylcysteine.

We have previously demonstrated that nitroso-chloramphenicol (NO-CAP) in small concentrations causes the hydrolysis of isolated double stranded DNA in vitro and this action is blocked by sulfhydryl groups. The present study was designed to assess damage to isolated DNA as well as intact cells DNA and examine the protective effect of N-acetylcysteine (NAC). Using alkaline sucrose gradient sedimentation and the alkali elution technique of Kohn we were able to demonstrate DNA damage in Raji cells as well as phytohemagglutinin stimulated human lymphocytes after exposure to NO-CAP. Damage could be totally blocked by NAC. In preliminary studies we also observed that NAC protects bone marrow cells from the growth-inhibitory effects of chloramphenicol and thiamphenicol.

Sensitivity of cultured pancreatic carcinoma cells to Acinetobacter glutaminase-asparaginase.

Cultured human pancreatic carcinoma cells (MIA PaCa-2) have been shown previously to be very sensitive to E. coli L-asparaginase (EC II). The present studies have demonstrated that another enzyme, Acinetobacter glutaminase-asparaginase (AGA) is much more effective in inhibiting cell growth. At the concentration of 0.0025 U/ml of AGA activity the enzyme totally inhibited cell growth, whereas the EC II with the same concentration did not show any effect. The inhibition of cell growth correlated well with inhibition of protein and glycoprotein synthesis. The addition of L-glutamine at the concentration of 1 mM completely reversed the inhibition of protein synthesis. Similarly, the addition of L-glutamine at the concentration of 3 mM daily on 3 successive days after adding AGA resulted in significant reversal of growth inhibition. The results of this study indicate that the action of AGA on MIA PaCa-2 is, to a great extent, exerted through its L-glutaminase activity.

Nitroso-chloramphenicol: possible mediator in chloramphenicol-induced aplastic anemia.

We compared the effects of CAP and its nitroso derivative on DNA synthesis, CFU-C growth, and cell viability in human bone marrow and on mouse CFU-S viability in vitro. As previously reported from this laboratory, CAP inhibited DNA synthesis only when used in high concentrations (greater than 3 x 10(-4)M), and the inhibition was largely reversible. It also caused a reversible concentration-dependent inhibition of CFU-C growth but did not affect marrow cell viability. In sharp contrast, nitroso-CAP inhibited DNA synthesis in much lower concentrations and caused irreversible inhibition of CFU-C growth and cell death at 5 x 10(-5)M as well as irreversible mouse CFU-S damage. In a rapidly growing human lymphoid cell line, nitroso-CAP caused accumulation of cells in the G2M phase and increasing cell death within the arrested population. We have postulated from these and other observations that CAP-induced aplastic anemia occurs in the predisposed host who provides the milieu for the transformation of the p-NO2 group of the drug to toxic intermediates.

Mechanism of sensitivity of cultured pancreatic carcinoma to asparaginase.

The effects of E. coli L-asparaginase on cultured human pancreatic carcinoma (MIA PaCa-2) have been studied. The enzyme (1 U/ml) inhibited growth and protein synthesis in both MIA PaCa-2 and PANC-1, another pancreatic carcinoma cell line, but had little or no effect on human breast carcinoma or melanoma cells. The inhibition of protein synthesis by E. coli L-asparaginase was largely reversed by L-glutamine but not by L-asparagine. The growth of both MIA PaCa-2 and PANC-1 showed absolute dependence on L-glutamine. These results indicate that the effect of E. coli L-asparaginase on cultured pancreatic carcinoma cells is exerted at least in part through its L-glutaminase activity. Although the addition of L-glutamine to the culture appeared to prevent cell death caused by L-asparaginase, it did not restore the ability of the cells to proliferate. Asparaginase derived from vibrio succinogenes, which is virtually free of L-glutaminase activity, was equally inhibitory to MIA PaCa-2 cell growth but did not affect protein synthesis. It is concluded that the inhibition of growth of cultured pancreatic carcinoma cells by E. coli asparaginase is a combined function of both its L-asparaginase and L-glutaminase activity.

In vitro evidence for genetically determined variations in marrow erythroid cell sensitivity to chloramphenicol.

The effect of chloramphenicol (CAP) was examined on the in vitro growth of CFU-E and CFU-C from four strains of mice: C57L/J, A/HEJ and their first generation hybrid LAF1, and the inbred strain C57BL/6J. The growth of marrow CFU-E from C57L/J and the hybrid LAF1 was inhibited by 82--96% at a CAP concentration of 10 microgram/ml compared to only 22--39% for CFU-E from A/HEJ and C57BL/6J. There was little difference in the degree of inhibition of CFU-C growth by CAP among the four strains, with that of C57L/J showing a slightly greater sensitivity to the drug. These results indicate a genetically-determined variation in the sensitivity of marrow erythroid cells to CAP.

Purification and characterization of a plasminogen activator secreted by cultured human pancreatic carcinoma cells.

A plasminogen activator secreted by cultured human pancreatic carcinoma (Mia PaCa-2) cells has been purified to apparent homogeneity by procedures including Sepharose-L-arginine methyl ester affinity chromatography, Sephadex G-200 gel filtration, isoelectric focusing, and sodium dodecyl sulfate gel electrophoresis. The plasminogen activator shares many properties with urokinase including: molecular weight (55 000), isoelectric point (8.7), heat stability (60 degrees C, 30 min), PH stability (1.5-10), and its mode of activation of plasminogen. The intracellular enzyme is membrane bound and can be solubilized by detergent. Solubilized activator has a molecular weight similar to that of the secreted enzyme as determined by sodium dodecyl sulfate gel electrophoresis. The production of plasminogen activator by Mia PaCa-2 cells is totally inhibited by actinomycin D and cycloheximide.

Human pancreatic carcinoma (MIA PaCa-2) in continuous culture: sensitivity to asparaginase.

An undifferentiated human pancreatic carcinoma has been established in continuous culture and is grown in Dulbecco's modified. Eagle's medium fortified with 10% fetal calf serum and 2.5% horse serum. The established cell line (MIA PaCa-2) has a doubling time of 40 h. The cells are large with abundant cytoplasm, exhibit a high degree of aneuploidy and have a tendency to grow on top of other cells. MIA PaCa-2 grows in soft agar with a colony-forming efficiency of 19%. Both MIA PaCa-2 cells and a cell line from another pancreatic carcinoma obtained from National Cancer Institute (NCI) are sensitive to asparaginase, a property not shared by several other human tumor cell lines tested.

Characteristics of rat carcinoma in culture.

Rat chloroma cells have been propagated in permanent suspension cultures and are grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum and 2.5% horse serum. Several lines have been established; the one of longest duration, Mia C51, has been maintained for over 18 months and has undergone over 100 transfers. Mia C51 cells have a doubling time of 12 hr and maintain many of the properties of the parent tumor, including the characteristic greenish color with high myeloperoxidase activity, an an aneuploid chromosomal pattern, and intact tumorigenicity. They will uniformly produce greenish chloroma tumors when injected into newborn rats. Electron microscopic examination of chloroma tumors and the cultured cells derived from them reveal the presence of extracellular mature and immature type C virus particles morphologically typical of oncornaviruses. Chloroma cells obtained from tumors that lost their alkaline phosphatase activity after repeated transfer regain full activity in culture. Studies using the antigen-antibody crossed electrophoresis indicate that the loss of alkaline phosphatase activity represents a true decrease in alkaline phosphatase protein, which is restored under culture conditions. The availability of a permanent chloroma cell line in culture that maintains the biological properties of the parent tumor provides a useful model for the study of myeloid leukemia.