28-day repeated dose oral toxicity of recombinant human holo-lactoferrin in rats.

Recombinant human holo-lactoferrin (holo-rhLF) was orally administered, via gavage, to Wistar rats at 1000, 500 and 100mg/kgbw/day for 28 days. The test article, holo-rhLF, was expressed in rice grain, extracted, purified and saturated with iron. During the 28-day period, animals were examined for evidence of toxicity. On day 29, the animals were exsanguinated, examined for gross pathology, and tissues preserved for histopathology. There were no deaths caused by holo-rhLF and in-life physical signs were generally normal. Although statistical differences were noted in some hematology, clinical chemistry and heart/body weight ratios, they were of questionable biological significance. A significantly greater total iron binding capacity (TIBC) was detected in the blood of male animals dosed with holo-rhLF. Serum was analyzed for the presence of IgG and IgE antibodies; demonstrating low levels of IgG antibodies to the human protein, but no increase in IgE antibodies. There was no increase in serum lactoferrin levels. The results of the 28-day oral administration demonstrate a lack of toxicity of holo-rhLF in rats. There were no treatment related, toxicologically relevant changes in clinical signs, growth, food consumption, hematology, clinical chemistry, organ weights or pathology. The no observed adverse effect level (NOAEL) is greater than 1000 mg/kg/day.

28-Day repeated dose oral toxicity of recombinant human apo-lactoferrin or recombinant human lysozyme in rats.

Lactoferrin and lysozyme are important proteins of the human innate immune system. These proteins are found in breast milk and have been associated with improved infant health. Recombinant human apo-lactoferrin (apo-rhLF), 1800 and 180mg/kg bw/day, and recombinant human lysozyme (rhLZ), 360 and 36mg/kg bw/day, were orally administered to Wistar rats for 28 days. Apo-rhLF and rhLZ were expressed in rice grain, extracted, purified; the lactoferrin was iron desaturated. The animals were examined for evidence of toxicity; there were no deaths and in-life physical signs were normal. Transient differences in mean food consumption occurred in high dose apo-rhLF and low dose LZ females at week three. There were no biologically significant differences in hematological or clinical chemistry parameters. Necropsy results were normal and microscopic evaluation showed no treatment related changes in animals dosed with 1800mg/kg/day apo-rhLF or 360mg/kg/day rhLZ. The results of the 28-day oral administration demonstrate a lack of toxicity of apo-rhLF and rhLZ in rats. There were no treatment related, toxicologically relevant changes in clinical signs, growth, food consumption, hematology, clinical chemistry, organ weight and pathology. The no observed adverse effect level (NOAEL) is greater than 1800mg/kg/day for apo-rhLF and 360mg/kg/day for rhLZ.

Acetaminophen-induced proliferation of breast cancer cells involves estrogen receptors.

Previous studies have shown that acetaminophen, a common analgesic/antipyretic, induces proliferation of cultured breast cancer cells containing both estrogen and progesterone receptors (ER+/PR+). The main objective of this study was to evaluate the involvement of ERs in this effect. First, the effects of therapeutic acetaminophen concentrations were compared in breast cancer cells with high ERs and in T47Dco cells with lower ERs, to determine if acetaminophen-induced proliferation depends on ER levels. Second, the effects of two antiestrogens (ICI 182,780 and 4'-hydroxytamoxifen) on acetaminophen-induced proliferation were determined in three human breast cancer cell lines: two ER+/PR+ (MCF7, T47D) and one ER-/PR- (MDA-MB-231). Third, ER binding assays were performed in MCF7 cells to determine if acetaminophen competed with estradiol for binding to ERs. Proliferation endpoints monitored included percent cells in the DNA synthesis phase of the cell cycle, 3H-thymidine incorporation into DNA, and cell number. Acetaminophen did not induce DNA synthesis in T47Dco cells, but did in cells with higher ER levels, suggesting high ER levels are necessary for acetaminophen to induce proliferation. Antiestrogens inhibited acetaminophen-induced proliferation in ER+/PR+ cells while no effects were observed in ER-/PR- cells, further supporting ER involvement. However, acetaminophen did not compete with estradiol for binding to ERs in ER+/PR+ cells. Collectively, these data suggest that acetaminophen induces breast cancer cell proliferation via ERs without binding to ERs like estradiol. The second purpose of this study was to determine if acetaminophen is estrogenic/antiestrogenic in vivo (uterotrophic assays). Acetaminophen has no antiestrogenic/estrogenic activity in mice or rats uteri.

Suppression of anti-microtubule agent-induced apoptosis by nitric oxide: possible mechanism of a new drug resistance.

The propensity of a cell to undergo apoptosis has been proposed to be a determinant of sensitivity to anti-microtubule agents. The anti-microtubule agents vincristine and paclitaxel induce key features of apoptosis, such as intranucleosomal DNA fragmentation and changes in nuclear morphology in the human neuroblastoma cell line, NB-39-nu. Nitric oxide (NO) generated from NO-releasing drugs prevented anti-microtubule agent-induced apoptosis in this cell line. The mechanism of suppression of apoptosis by NO appears to be via the inhibition of an interleukin-1beta converting enzyme-like protease cascade. This finding reveals a new biological function of NO, as well as a new molecular insight into resistance to chemotherapy with anti-microtubule agents.

Role of nitric oxide in acetaminophen-induced hepatotoxicity in the rat.

Acetaminophen is a mild analgesic and antipyretic agent known to cause centrilobular hepatic necrosis at toxic doses. Although this may be due to a direct interaction of reactive acetaminophen metabolites with hepatocyte proteins, recent studies have suggested that cytotoxic mediators produced by parenchymal and nonparenchymal cells also contribute to the pathophysiological process. Nitric oxide is a highly reactive oxidant produced in the liver in response to inflammatory mediators. In the present studies we evaluated the role of nitric oxide in the pathophysiology of acetaminophen-induced liver injury. Treatment of male Long Evans Hooded rats with acetaminophen (1 g/kg) resulted in damage to centrilobular regions of the liver and increases in serum transaminase levels, which were evident within 6 hours of treatment of the animals and reached a maximum at 24 hours. This was correlated with expression of inducible nitric oxide synthase (iNOS) protein in these regions. Hepatocytes isolated from both control and acetaminophen-treated rats were found to readily synthesize nitric oxide in response to inflammatory stimuli. Cells isolated from acetaminophen-treated rats produced more nitric oxide than cells from control animals. Production of nitric oxide by cells from both control and acetaminophen-treated rats was blocked by aminoguanidine, a relatively specific inhibitor of iNOS. Arginine uptake and metabolism studies revealed that the inhibitory effects of aminoguanidine were due predominantly to inhibition of iNOS enzyme activity. Pretreatment of rats with aminoguanidine was found to prevent acetaminophen-induced hepatic necrosis and increases in serum transaminase levels. This was associated with reduced nitric oxide production by hepatocytes. Inhibition of toxicity was not due to alterations in acetaminophen metabolism since aminoguanidine had no effect on hepatocyte cytochrome P4502E1 protein expression or N-acetyl-p-benzoquinone-imine formation. Taken together, these data demonstrate that nitric oxide is an important mediator of acetaminophen-induced hepatotoxicity.

Arginine metabolism in keratinocytes and macrophages during nitric oxide biosynthesis: multiple modes of action of nitric oxide synthase inhibitors.

Nitric oxide is an important cellular mediator produced in keratinocytes and macrophages from arginine by the enzyme nitric oxide synthase during inflammatory reactions in the skin. We found that gamma-interferon stimulated nitric oxide production and the expression of inducible nitric oxide synthase in both cell types. However, macrophages produced more nitric oxide and nitric oxide synthase protein, and at earlier times than keratinocytes. Keratinocytes treated with gamma-interferon took up more arginine than macrophages; however, they were less efficient in metabolizing this amino acid and exhibited reduced nitric oxide synthase enzyme activity. In both cell types, the nitric oxide synthase inhibitors, N(G)-monomethyl-L-arginine (NMMA), L-N5-(iminoethyl)ornithine, L-canavanine, and N(omega)-nitro-L-arginine, as well as lysine, ornithine, and homoarginine markedly reduced arginine uptake. In contrast, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine benzyl ester were poor inhibitors of arginine uptake, while aminoguanidine had no effect on uptake of arginine by the cells. Moreover, NMMA was found to inhibit simultaneously arginine uptake and nitric oxide synthase enzyme activity in both cell types, whereas aminoguanidine only affected nitric oxide synthase activity. No major differences were observed between keratinocytes and macrophages. Taken together, these data demonstrate that, although keratinocytes and macrophages both synthesize nitric oxide, its production is regulated distinctly in these two cell types. Furthermore, in these cells, nitric oxide synthase inhibitors such as NMMA exhibit at least two sites of action: inhibition of nitric oxide synthase and cellular uptake of arginine.

5-Fluorouracil suppresses nitric oxide biosynthesis in colon carcinoma cells.

Nitric oxide is an important cellular mediator that plays a role in regulating cellular proliferation of both normal and tumor cells. In the present study, we characterized nitric oxide production by the human colon adenocarcinoma cell line DLD-1 and examined the effects of 5-fluorouracil (5-FUra), an antimetabolite effective against colon tumors, on nitric oxide production. IFN-gamma was found to be a potent inducer of nitric oxide production in DLD-1 cells. This effect was dependent on L-arginine and blocked by the nitric oxide synthase inhibitors NG-monomethyl-L-arginine, nitroarginine, and aminoguanidine. Production of nitric oxide by DLD-1 cells was due to the expression of the inducible (type II) form of nitric oxide synthase. mRNA for the nitric oxide synthase was present in both untreated and IFN-gamma-stimulated cells, as determined by RT-PCR, suggesting that expression of enzyme is regulated posttranscriptionally. Treatment of DLD-1 cells with concentrations of 5-FUra that are not growth inhibitory or cytotoxic strongly inhibited their ability to express nitric oxide synthase and produce nitric oxide in response to IFN-gamma. This effect was not reversed with thymidine, indicating that inhibition of nitric oxide production was due to incorporation of 5-FUra into RNA. However, pretreatment of DLD-1 cells with 5-FUra before stimulation with IFN-gamma also suppressed nitric oxide production. Thus, inhibition of nitric oxide production was not due directly to incorporation of 5-FUra into the mRNA for nitric oxide synthase. Taken together, these data suggest that inhibition of nitric oxide biosynthesis in colon tumor cells by 5-FUra may underlie, at least in part, the efficacy of this antitumor agent.