Effect of dietary conjugated linoleic acid supplementation on the technological quality of backfat of pigs.

Pigs were fed diets containing 0, 0.25, 0.5 and 1% conjugated linoleic acid (CLA). Compared to controls, backfat from CLA fed pigs was firmer and extracted lipid contained increasing amounts of CLA, but a ±11% overall decrease in unsaturated fatty acids and a ±5% overall increase in each of C16:0 and C18:0 saturated fatty acids were noted. This resulted in a change in the melting properties of fat. The onset setting temperature increased from ±14°C to ±18°C for lipid of backfat of pigs from the 0.25 and 0.5% CLA supplementation groups, and to ±26°C for lipid from the 1% CLA supplementation group. The final melting temperatures increased from ±37°C to ±43°C and ±45°C, respectively. The presence of ß'-crystals of C18:0-C16:0-C18:1c9 triacylglycerides in fat from CLA fed pigs and ß-crystals in fat from 1% CLA fed pigs was observed. Fatty acid and melting point results explained the improvement in the technological quality of backfat as a result of dietary CLA supplementation.

Cytotoxicity and cell death pathways invoked by two new rhodium-ferrocene complexes in benign and malignant prostatic cell lines.

BACKGROUND: Apoptotic propensity is currently viewed as an important parameter in drug-induced toxicity. But other cell death pathways exist e.g. micronucleation, intermitotic cell death, abnormal nuclear morphology and necrosis. This investigation explores the onset of apoptosis and abnormal morphology in response to 3 drugs i.e. Cisplatin, a novel Ferrocene (fctfa) and a novel Rhodium-Ferrocene [Rh(fctfa)(cod)] complex. MATERIALS AND METHODS: A pair of prostate cell lines from normal human prostate epithelium (1542N) and malignant human prostate epithelium (1542T) were exposed to increasing concentrations of the drugs for 24 hours, double-stained with FITC-Annexin V and with Propidium Iodide and analysed by dual parameter flow cytometry to quantitate viable cells in quadrant I, early apoptotic cells in quadrant IV and late apoptotic/necrotic cells in quadrant III. Apoptosis was also scored by microscopy after Acridine Orange staining, by Western blots for caspase 3 induction and for caspase 8 induction using a colorimetric assay. RESULTS: The toxicity of Cisplatin and the Ferrocene and Rhodium-Ferrocene complexes was found to be 0.9-1.3 microM; 4.1-4.5 microM and 10.1-13.2 microM, respectively. Apoptotic propensity scored after 24 hours was found to be dose-dependent and in the range of 7-19% for Cisplatin and 1-4.1% for the Ferrocene and Rhodium-Ferrocene complexes. Cisplatin produces a distinct apoptotic response followed by a necrotic response, whereas the Ferrocene and the Rhodium-Ferrocene complexes produce a massive necrotic reaction in the region of 3-19% and very little if any apoptosis. Absence of apoptosis was corroborated by lack of caspase 3 activation, absence of typical apoptotic morphology and by lack of caspase 8 activation. CONCLUSION: The 3 drugs Cisplatin, the novel Ferrocene and the novel Rhodium-Ferrocene complexes show similar toxicities in the 1-10 micro-molar range in prostate cell lines. However the drugs differ significantly in the activation of death pathways. While Cisplatin predominantly induces apoptosis documented by morphology, Annexin V staining and caspase 8 activation, the Ferrocene and Rhodium-Ferrocene complexes induce late necrosis and abnormal nuclear morphology. Unlike Cisplatin-treated cells which enter apoptosis and necrosis sequentially, the 2 Ferrocene drugs invoke direct entry of cells into late necrosis without first entering the early apoptotic compartment.

Polyaspartamides as water-soluble drug carriers. Part 1: Antineoplastic activity of ferrocene-containing polyaspartamide conjugates.

BACKGROUND: Numerous detrimental side effects and/or lack of water-solubility of anticancer drugs often prove dose-limiting in chemotherapy. Water-soluble polymeric drug carriers may overcome/minimise many of these limitations. MATERIALS AND METHODS: Aspartic acid polymers to which ferrocene-containing antineoplastic agents are covalently bound, were tested for cytotoxicity against murine EMT-6 cancer cells. Cell survival was measured by means of the colorometric 3-(4,5-dimethylthiazol-2-yl)-diphenyltertrazolium bromide assay. RESULTS: The 90% lethal dosage of pure 3-ferrocenylbutanoic acid is 452 microg/mL LD90 for the polymeric derivative, expressed in terms of 3-ferrocenylbutanoic acid content, is only 65 microg/ml. A polymer structural effect in drug activity was evident: longer side chains linking drugs to polymer backbones enhanced drug activity. Drug activity is also enhanced if drug modifications (to enable drug anchoring) resulted in a lower ferrocenyl reduction potential. CONCLUSIONS: The effectivity of antineoplastic drugs may be enhanced by covalently anchoring them on suitable biodegradable water-soluble polymeric drug carriers.

Chelator effect on ion diffusion in ferrous-sulfate-doped gelatin gel dosimeters as analyzed by MRI.

Ferrous-sulfate-doped gelatin gel dosimeters are useful tools for the measurement of three-dimensional absorbed radiation dose distributions. The diffusion of ferric ions through these gels causes degradation with time of the dose distribution image. It would be useful to reduce ferric ion diffusion without decreasing gel sensitivity. The amount of ferric ion diffusion is a function of the time delay after radiation, the gel temperature, and the gel concentration. These effects can be quantified by measuring the ferric ion diffusion coefficient. Determination of the diffusion coefficient by irradiating the lower section of a cylinder of gel, which was then imaged repeatedly over time with a clinical magnetic resonance imager, is described. Analysis of the edge spread function formed at each of several times after irradiation by drawing a profile over the imaged junction between the irradiated and unirradiated halves of the cylinder, gave estimates of the variance of the edge spread function. These variances were used to obtain an estimate of the ferric ion diffusion coefficient for the gel. A method of reducing ferric ion diffusion by adding a chelator and the cross linkage agent formaldehyde is suggested. The chelators investigated were 1,10 phenanthroline, xylenol orange, and bathophenanthroline disulfonic acid. These reduced diffusion to varying extents, and influenced the gel sensitivity. The diffusion coefficient in gels containing xylenol orange was found to be 0.44 mm2h-1. The gel sensitivity was 0.0093 s-1Gy-1. This compared with a diffusion coefficient of 0.82 mm2h-1 for the base line gel that did not contain formaldehyde or chelators. The sensitivity of this base line gel was 0.0129 s-1Gy-1. The addition of xylenol orange produced the most improved gel dosimeter of the gels studied. This gel had a decreased ferric ion diffusion coefficient and a decreased sensitivity. It was still sensitive enough to be useful.

Kinetic studies on the reduction of the tyrosyl radical of the R2 subunit of E. coli ribonucleotide reductase.

Kinetic studies at 25 degrees C, I = 0.100 M (NaCl), on the reduction of the tyrosyl radical of the R2 protein of E. coli ribonucleotide reductase with hydroxyurea (HU), N-methylhydroxylamine, catechol, and seven hydroxamic acid derivatives are reported. There are no pH-dependences in the range 6.2-8.6 investigated except that introduced with N-methylhydroxylamine which itself protonates in this range. At pH 7.6 the rate constant (0.46 M-1 s-1) for the HU reaction is in agreement with earlier values. Slower reactions are observed with the bulkier acetohydroxamic (0.020 M-1 s-1) and benzohydroxamic acids (0.040 M-1 s-1). In the case of N-methylhydroxylamine the rate constant (0.41 M-1 s-1 at pH 7.6) decreases with pH, and it is concluded that the protonated form CH3NH2+OH(pKa = 6.2) has little or no reactivity with Tyr. For this reaction under air-free conditions a second-stage (0.027 M-1 s-1) corresponding to reduction of Fe(III)2 is observed. Mid-point redox potentials for the reductants and estimates of reduction potentials applying in the case of the protein are considered. The reactions with 1,2-dihydroxybenzene (catechol) and 3,4-dihydroxybenzohydroxamic acid (Didox) also have two stages, when the initial Tyr reduction, rate constants/M-1 s-1 for catechol (3.2) and Didox (0.010), is followed by removal of the Fe(III) to give catechol and catechol like Fe(III)-complexed products. The single stage reactions of the hydroxamic acid derivatives which incorporate charged amino-acid groups L-glutamic acid, L-histidine, L-glycine and L-lysine, are slow, and saturation kinetics are observed consistent with association (small K values) prior to redox. The mechanism of reduction of R2-Tyr by all of the reagents studied is discussed.

Towards an understanding of the reactivity of E. coli R2 ribonucleotide reductase: a mechanistic approach to inactivation.

Five categories of reaction of the Escherichia coli R2 protein of ribonucleotide reductase (RNR) are defined from mechanistic studies with hydroxyurea, methylhydroxylamine, hydroxamic acid derivatives, long-lived organic radicals of which methyl viologen MV+ is a good example, hydrazine, catechol and their derivatives. Attention is focused on whether a particular reagent reduces only the tyrosyl radical (Tyr.) giving metR2, or the Tyr. and Fe(III)2 in consecutive steps to give fully reduced R2. In the case of hydrazine (N2H4), reduction of both the Tyr and Fe(III)2 occurs in a uniphasic process, while with di-imide (N2H2) it has already been demonstrated that the Tyr. is reduced and that Fe(II)Fe(III) semi-metR2 is formed. A further mechanism is observed with catechol and catechol-like derivatives (in this work Didox), in which there is reduction of the Tyr. in the first stage, followed by scavenging of the Fe(III) in the second. The latter offers a more permanent inactivation of R2, meriting more extensive study in the context of cancer drug therapy. Comparative studies on mouse R2 suggest that the Tyr. and Fe(III)2 of mammalian R2 forms may be more exposed, and as compared to E. coli R2 are approximately an order of magnitude more reactive.