Porphyrin Accumulation and Biliary Lithiasis Causing Diffusely Black Livers in Broiler Chickens.

Two 7-wk-old broiler chickens presented with uniformly black livers upon postslaughter examination, while all other organs as well as their carcasses were grossly normal. No clinical signs were reported by the field veterinarian prior to slaughter. Other broiler chickens within the same flock were unaffected. Microscopically, the liver exhibited variably sized, globoid concrements that were dark brown to green-brown and birefringent under polarized light. Ultrastructurally, concrements consisted of radially arranged electron-dense crystal spicules. Concrements were located in hepatocytes, within ecstatic bile canaliculi, or surrounded by small clusters of macrophages. Liquid chromatography assay determined the presence of protoporphyrin IX in the affected liver.Two 7-wk-old broiler chickens presented with uniformly black livers upon postslaughter examination, while all other organs as well as their carcasses were grossly normal. No clinical signs were reported by the field veterinarian prior to slaughter. Other broiler chickens within the same flock were unaffected. Microscopically, the liver exhibited variably sized, globoid concrements that were dark brown to green-brown and birefringent under polarized light. Ultrastructurally, concrements consisted of radially arranged electron-dense crystal spicules. Concrements were located in hepatocytes, within ecstatic bile canaliculi, or surrounded by small clusters of macrophages. Liquid chromatography assay determined the presence of protoporphyrin IX in the affected liver.

Vivid biofluorescence discovered in the nocturnal Springhare (Pedetidae).

Biofluorescence has been detected in several nocturnal-crepuscular organisms from invertebrates to birds and mammals. Biofluorescence in mammals has been detected across the phylogeny, including the monotreme duck-billed platypus (Ornithorhyncus anatinus), marsupial opossums (Didelphidae), and New World placental flying squirrels (Gluacomys spp.). Here, we document vivid biofluorescence of springhare (Pedetidae) in both museum specimens and captive individuals-the first documented biofluorescence of an Old World placental mammal. We explore the variation in biofluorescence across our sample and characterize its physical and chemical properties. The striking visual patterning and intensity of color shift was unique relative to biofluorescence found in other mammals. We establish that biofluorescence in springhare likely originates within the cuticle of the hair fiber and emanates, at least partially, from several fluorescent porphyrins and potentially one unassigned molecule absent from our standard porphyrin mixture. This discovery further supports the hypothesis that biofluorescence may be ecologically important for nocturnal-crepuscular mammals and suggests that it may be more broadly distributed throughout Mammalia than previously thought.

5-Aminolevulinate dehydratase porphyria: Update on hepatic 5-aminolevulinic acid synthase induction and long-term response to hemin.

BACKGROUND: 5-Aminolevulinic acid dehydratase (ALAD) porphyria (ADP) is an ultrarare autosomal recessive disease, with only eight documented cases, all of whom were males. Although classified as an acute hepatic porphyria (AHP), induction of the rate limiting hepatic enzyme 5-aminolevulinic acid synthase-1 (ALAS1) has not been demonstrated, and the marrow may also contribute excess 5-aminolevulinic acid (ALA). Two patients have died and reported follow up for the others is limited, so the natural history of this disease is poorly understood and treatment experience limited. METHODS: We report new molecular findings and update the clinical course and treatment of the sixth reported ADP patient, now 31 years old and the only known case in the Americas, and review published data regarding genotype-phenotype correlation and treatment. RESULTS: Circulating hepatic 5-aminolevulinic acid synthase-1 (ALAS1) mRNA was elevated in this case, as in other AHPs. Gain of function mutation of erythroid specific ALAS2 - an X-linked modifying gene in some other porphyrias - was not found. Seven reported ADP cases had compound heterozygous ALAD mutations resulting in very low residual ALAD activity and symptoms early in life or adolescence. One adult with a germline ALAD mutant allele developed ADP in association with a clonal myeloproliferative disorder, polycythemia vera. CONCLUSIONS: Elevation in circulating hepatic ALAS1 and response to treatment with hemin indicate that the liver is an important source of excess ALA in ADP, although the marrow may also contribute. Intravenous hemin was effective in most reported cases for treatment and prevention of acute attacks of neurological symptoms.

Cystathionine-γ-lyase (CSE) deficiency increases erythropoiesis and promotes mitochondrial electron transport via the upregulation of coproporphyrinogen III oxidase and consequent stimulation of heme biosynthesis.

BACKGROUND: Hydrogen sulfide (H2S) is an endogenous gasotransmitter produced by mammalian cells. The current study investigated the potential role of H2S in the regulation of heme biosynthesis using mice deficient in cystathionine gamma-lyase (CSE), one of the three major mammalian H2S-producing enzymes. METHODS: Wild-type and global CSE-/- mice, as well as mitochondria prepared from their liver were used. In vivo, arterial and venous blood gases were measured, and survival of the mice to severe global hypoxia was monitored. Ex vivo, expression of various heme biosynthetic enzymes including coproporphyrinogen oxidase (CPOX) was measured, and mitochondrial function was evaluated using Extracellular Flux Analysis. Urine samples were collected to measure the oxidized porphyrinogen intermediates. The in vivo/ex vivo studies were complemented with mitochondrial bioenergetic studies in hepatocytes in vitro. Moreover, the potential effect of H2S on the CPOX promoter was studied in cells expressing a CPOX promoter construct system. RESULTS: The main findings are as follows: (1) CSE-/- mice exhibit elevated red blood cell counts and red blood cell mean corpuscular volumes compared to wild-type mice; (2) these changes are associated with elevated plasma and liver heme levels and (3) these alterations are likely due to an induction of CPOX (the sixth enzyme involved in heme biosynthesis) in CSE-/- mice. (4) Based on in vitro promoter data the promoter activation of CPOX is directly influenced by H2S, the product of CSE. With respect to the potential functional relevance of these findings, (5) the increased circulating red blood cell numbers do not correspond to any detectable alterations in blood gas parameters under resting conditions, (6) nor do they affect the hypoxia tolerance of the animals in an acute severe hypoxia model. However, there may be a functional interaction between the CSE system and the CPOX system in terms of mitochondrial bioenergetics: (7) CSE-/- hepatocytes and mitochondria isolated from them exhibit increased oxidative phosphorylation parameters, and (8) this increase is partially blunted after CPOX silencing. Although heme is essential for the biosynthesis of mitochondrial electron chain complexes, and CPOX is required for heme biosynthesis, (9) the observed functional mitochondrial alterations are not associated with detectable changes in mitochondrial electron transport chain protein expression. CONCLUSIONS: The CSE system regulates the expression of CPOX and consequent heme synthesis. These effects in turn, do not influence global oxygen transport parameters, but may regulate mitochondrial electron transport.

Harderoporphyria: Case of lifelong photosensitivity associated with compound heterozygous coproporphyrinogen oxidase (CPOX) mutations.

A 78-year-old man with a history of neonatal anemia and jaundice and life-long photosensitivity was found to have harderoporphyria, as evidenced by increased porphyrins in urine, plasma, erythrocytes and feces including large amounts of harderoporphyrin in feces and erythrocytes. Two previously undescribed coproporphyrinogen oxidase (CPOX) mutations were identified, including a deletion of four amino acids in a region of the enzyme mutated in 7 of the 8 previously reported cases. This case increases the molecular heterogeneity of this rare porphyria, and illustrates that it should be considered as a cause of chronic photosensitivity and porphyrin elevation at any age.

Novel effects of phytoestrogenic soy isoflavones on serum calcium and chloride in premenopausal women: A 2-year double-blind, randomized, placebo-controlled study.

BACKGROUND: Soy phytoestrogens are potential alternatives to postmenopausal hormone replacement therapy (HRT). Adverse effects of HRT such as myocardial infarction, stroke, and pulmonary embolism are mediated by calcium-induced signaling. OBJECTIVE: To determine whether soy isoflavones affect serum calcium in healthy female subjects. DESIGN: In a double-blind trial, 197 premenopausal women were randomly assigned to either isoflavone (N = 99) or placebo pills (N = 98) 5 days per week for up to 2 years, plus prenatal vitamins. Isoflavone pills contained 60 mg genistein, 60 mg daidzein and 16.6 mg glycitein (expressed as aglycone equivalents). All pills contained 15 mg riboflavin as an adherence marker. Blood chemistries and urinary daidzein, genistein and riboflavin were measured multiple times during the luteal phase before and during treatment. RESULTS: Analysis of the adherent population (N = 83 per group), revealed significantly strong associations between urinary levels of isoflavones and serum concentrations of calcium (regression coefficients 0.082 for daidzein and 0.229 for genistein, all P < 0.01) and chloride (regression coefficient, -1.537 for genistein, P < 0.0001), mediated in part by albumin. The effects amounted to mean changes of +0.24 mg/dL for calcium and -1.45 mEq/L for chloride, with each visit for subjects excreting the most vs. the least amounts of isoflavones. These associations were not evident in the intention-to-treat analysis (N = 197) that did not assess expected variations in isoflavone levels within and between subjects from metabolism and adherence. CONCLUSIONS: These novel and strong effects of soy isoflavones on calcium homeostasis have important implications for long term effects of these natural substances on cardiovascular diseases.

Riboflavin as an independent and accurate biomarker for adherence in a randomized double-blind and placebo-controlled clinical trial.

BACKGROUND: Medication adherence is critical for success of clinical trials. OBJECTIVE: To assess oral riboflavin is an adherence marker. METHODS: Riboflavin was incorporated into active treatment and placebo pills for a clinical trial lasting for 2 years. RESULTS: The accuracy (area under the receiver operating curve) of urinary riboflavin was 0.91 as a binary classifier of adherence, and was similar or better than for two active study ingredients daidzein (0.92) and genistein (0.87) (all p < 0.0001). Decreased adherence over time was similar in the two study groups. CONCLUSION: Riboflavin is an accurate and useful biomarker for study pill ingestion.

Riboflavin as an oral tracer for monitoring compliance in clinical research.

We studied urinary riboflavin as an objective biomarker of compliance in clinical research using a simplified method amenable to high throughput analysis. Six healthy women not taking vitamin supplements ingested a study pill containing riboflavin (32 mg) as an inactive tracer and the soy isoflavones daidzin (0.243 mmole) and genistin (0.222 mmole) as active ingredients once daily for four days. Riboflavin and metabolites of the isoflavones were measured in urine samples obtained before and after each pill. Urinary excretion of riboflavin and metabolites of both isoflavones peaked within 8 hrs and remained higher than baseline for 24 hrs. Urinary excretion of riboflavin was also measured in 152 additional women with unrestricted dietary supplement intakes. Mean and median urinary riboflavin concentrations in these women were 0.42 and 0.31 µg/mL, respectively, compared to 0.2 µg/mL during a riboflavin-restricted diet. Receiver operating characteristics (ROC) curves indicated that urinary riboflavin within 24 hrs after a 32 mg dose would perform well as a measure of compliance (all areas under the ROC curves ≥0.84. Samples collected during the initial 8 hrs after pill ingestion performed better as a compliance measure than later collections. In summary, compliance in a clinical study can be monitored in real time by incorporating 32 mg of riboflavin into study pills, with compliance indicated by urinary riboflavin levels increasing over individual baselines or to ≥1.0 µg/mL, with a false positive rate of being classified as compliant at <5%.

Bioaccumulation and toxicity of gold nanoparticles after repeated administration in mice.

Gold nanoparticles (GNPs) offer a great promise in biomedicine. Currently, there is no data available regarding the accumulation of nanoparticles in vivo after repeated administration. The purpose of the present study was to evaluate the bioaccumulation and toxic effects of different doses (40, 200, and 400 microg/kg/day) of 12.5 nm GNPs upon intraperitoneal administration in mice every day for 8 days. The gold levels in blood did not increase with the dose administered, whereas in all the organs examined there was a proportional increase on gold, indicating efficient tissue uptake. Although brain was the organ containing the lowest quantity of injected GNPs, our data suggest that GNPs are able to cross the blood-brain barrier and accumulate in the neural tissue. Importantly, no evidence of toxicity was observed in any of the diverse studies performed, including survival, behavior, animal weight, organ morphology, blood biochemistry and tissue histology. The results indicate that tissue accumulation pattern of GNPs depend on the doses administered and the accumulation of the particles does not produce sub-acute physiological damage.

Up-regulation of heme oxygenase-1 in rat spleen after aniline exposure.

The splenic toxicity of aniline is characterized by vascular congestion, hyperplasia, fibrosis, and the development of a variety of sarcomas in rats. However, the underlying mechanisms by which aniline elicits splenotoxic response are not well understood. Previously we have shown that aniline exposure causes oxidative damage to the spleen. To further explore the oxidative mechanism of aniline toxicity, we evaluated the potential contribution of heme oxygenase-1 (HO-1), which catalyzes heme degradation and releases free iron. Male SD rats were given 1 mmol/kg/day aniline in water by gavage for 1, 4, or 7 days, and respective controls received water only. Aniline exposure led to significant increases in HO-1 mRNA expression in the spleen (2-and 2.4-fold at days 4 and 7, respectively) with corresponding increases in protein expression, as confirmed by ELISA and Western blot analysis. Furthermore, immunohistochemical assessment of spleen showed stronger immunostaining for HO-1 in the spleens of rats treated for 7 days, confined mainly to the red pulp areas. No changes were observed in mRNA and protein levels of HO-1 after 1 day exposure. The increase in HO-1 expression was associated with increases in total iron (2.4-and 2.7-fold), free iron (1.9-and 3.5-fold), and ferritin levels (1.9-and 2.1-fold) at 4 and 7 days of aniline exposure. Our data suggest that HO-1 up-regulation in aniline-induced splenic toxicity could be a contributing pro-oxidant mechanism, mediated through iron release, and leading to oxidative damage.

Zinc binding in pestivirus N(pro) is required for interferon regulatory factor 3 interaction and degradation.

Pestiviruses, such as bovine viral diarrhea virus and classical swine fever virus (CSFV), use the viral protein N(pro) to subvert host cell antiviral responses. N(pro) is the first protein encoded by the single large open reading frame of the pestivirus positive-sense RNA genome and has an autoproteolytic activity, cleaving itself off from the polyprotein. N(pro) also targets interferon regulatory factor 3 (IRF3), a transcription factor for alpha/beta interferon genes, and promotes its proteasomal degradation, a process that is independent of the proteolytic activity of N(pro). We determined that N(pro) contains a novel metal-binding TRASH motif consisting of Cys-X(21)-Cys-X(3)-Cys (where X is any amino acid) at its C-terminus. We also found that N(pro) coordinates a single zinc atom as determined by graphite furnace-atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry. Mutational and biochemical analyses show that the cysteine residues in the TRASH motif are required for zinc binding and protein stability. Individual substitutions of the cysteines in the TRASH motif of CSFV N(pro) abolished the interaction of N(pro) with IRF3 and resulted in the loss of virus-mediated IRF3 degradation in CSFV-infected cells. Thus, the zinc-binding ability of N(pro) in pestiviruses appears to be essential for the virus-mediated degradation of IRF3.

Human retinal cadmium accumulation as a factor in the etiology of age-related macular degeneration.

Cadmium is a naturally occurring, highly toxic, metallic element. It pollutes the environment as a result of industrial activity and accumulates in human tissues with a long biological half-life. Cadmium content has been demonstrated to increase in human retinal tissues as a function of age and tobacco smokers have approximately twice as much cadmium in retinal tissues than non-smokers. Smoking is also a key environmental risk factor for the retinal disease age-related macular degeneration (AMD). Recent studies have shown that urinary cadmium levels (a measure of Cd body burden) are higher in smokers who have AMD. We now report the Cd measurements in human retinal tissues from eyes afflicted with AMD compared to non-diseased eyes (controls) from age-matched donors. Human donor eyes frozen under argon gas were assessed for AMD severity using color stereoscopic fundus photographs and the Minnesota Grading System. Cadmium, zinc and, copper levels were measured in retinal tissues (neural retina, retinal pigment epithelium and choroid) using inductively coupled plasma mass spectrometry and graphite furnace spectrophotometry and values were normalized to tissue protein levels. Higher Cd levels were found in the neural retina and RPE for eyes afflicted with AMD compared to controls in males, differences were not statistically significant in females. The results indicate that higher retinal cadmium burdens are associated with the presence of AMD at least in males and suggest possible gender differences in the metabolism of metals in the human retina.

Zinc deficiency in Mexican American children: influence of zinc and other micronutrients on T cells, cytokines, and antiinflammatory plasma proteins.

BACKGROUND: The Third National Health and Nutrition Examination Survey suggested some Mexican American children are at risk of zinc deficiency. OBJECTIVE: We measured the effects of zinc and micronutrients or of micronutrients alone on indexes of cell-mediated immunity and antiinflammatory plasma proteins. DESIGN: Subjects (n = 54) aged 6-7 y were randomly assigned and treated in double-blind fashion in equal numbers with 20 mg Zn (as sulfate) and micronutrients or with micronutrients alone 5 d/wk for 10 wk. RESULTS: Before treatment the mean +/- SD plasma zinc was 14.9 +/- 1.7 micromol/dL and the range was within the reference; hair zinc was 1.78 +/- 0.52 micromol/g and 41.6% were < or =1.68 micromol/g; serum ferritin was 25.7 +/- 18.6 microg/L and 50.0% were < or =20 microg/L. The zinc and micronutrients treatment increased the lymphocyte ratios of CD4(+) to CD8(+) and of CD4(+)CD45RA(+) to CD4(+)CD45RO(+), increased the ex vivo generation of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), decreased the generation of interleukin-10 (IL-10), and increased plasma interleukin-1 receptor antagonist (sIL-1ra) and soluble tumor necrosis factor receptor 1 (sTNF-R1). Micronutrients alone increased the ratio of CD4(+) to CD8(+) but not of CD4(+)CD45RA(+) to CD4(+)CD45RO(+), increased IFN-gamma but had no effect on IL-2 or IL-10, and increased sIL-1ra but not sTNF-R1. Efficacy of zinc and micronutrients was greater than micronutrients alone for all indexes except the ratio of CD4(+) to CD8(+), which was affected similarly. CONCLUSIONS: Before treatment, concentrations of hair zinc in 41.6% of subjects and serum ferritin in 50% were consistent with the presence of zinc deficiency. The greater efficacy of the zinc and micronutrients treatment compared with micronutrients alone supports this interpretation.

Copper and zinc distribution in the human retina: relationship to cadmium accumulation, age, and gender.

The essential metals copper and zinc play vital roles in retinal cell survival and are crucial for the normal functioning of antioxidant enzymes. Retinal zinc deficiencies and decreased cellular antioxidative capacity have been linked to human retinal diseases including age-related macular degeneration (AMD). We recently reported that cadmium (a toxic metal with no known physiological function that interferes with copper and zinc metabolism) accumulates in human retinal tissues during aging. Moreover, cadmium content was higher in specific retinal tissues of aged women compared to men. Since cadmium, zinc and copper bind to similar proteins, we hypothesized that Cu and Zn content of human retinal tissues change as functions of cadmium accumulation during aging. Thus, we assessed the distribution of zinc and copper in the neural retina, retinal pigment epithelium (RPE) and choroid (Bruch's membrane-choroid; BMC) in male and female donors aged 1.5-87 years. Two independent methods, graphite furnace atomic absorption spectrometry and inductively-coupled plasma mass spectrometry, were used to measure Cd, Zn, and Cu in retinal tissues in human eyes from donors aged 1.5 to 87 years and the resulting values were normalized to protein concentration. Zn levels were approximately 5 times higher than Cu levels in the same tissues. The relative tissue distributions of these metals were: BMC>RPE>neural retina (Zn) and BMC>RPE=neural retina (Cu). In the choroid, mean Cu and Zn levels were higher in aged donors (>or=55 years old) than young donors (<55 years) and levels of these metals were strongly correlated with each other (r=0.90). In the neural retina, Cu and Zn both significantly decreased as a function of age. Several sex-related differences were found in the RPE. Specifically, copper levels were significantly higher in males than in females. In addition, both Zn and Cu levels in males were positively correlated with cadmium content, whereas this association did not occur in females. The results are consistent with co-regulation of zinc and copper stores in retinal tissues and suggest that the balance of these metals is associated with cadmium accumulation and gender. Thus, the roles of cadmium and gender differences in retinal metal balance warrant further investigation as factors in age-related retinal disease.

Cadmium accumulation in the human retina: effects of age, gender, and cellular toxicity.

Tobacco smoking and aging are among the few factors linked to age-related macular degeneration (AMD), a major cause of blindness in the elderly. Recent studies indicate that cadmium (Cd), an environmental toxic trace metal, is approximately four-fold higher in the retinas of smokers compared to non-smokers. In this study, we determined the effects of age and gender on Cd accumulation in human retinal tissues, specifically the neural retina, retinal pigment epithelium (RPE), and choroid. Cadmium levels in cultured RPE cells or retinal tissues isolated from frozen donor eyes were measured using inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrophotometry (GF-AAS). Cadmium uptake in cultured human RPE cells (ARPE-19) was also assessed using GF-AAS. Toxic effects of cadmium were determined from cell loss (measured as a decrease in cell density) and lactate dehydrogenase release (an indicator of membrane disruption). In "young" eyes (< 55 years) Cd was highest in the retinal pigment epithelium and lowest in the neural retina. Cd was higher in all tissues in aged eyes (>or=55 years) and was significantly higher in the neural retina and RPE in older females. Cultured RPE cells exposed to Cd showed altered cell morphology, decreased cell survival, elevated ROS levels and concentration-dependent disruption of membrane integrity. We conclude that cadmium is accumulated differently in the neural retinal and RPE of older men and women. The deleterious effects of Cd on RPE cells indicate that this environmental toxin is a potentially important factor in age-related retinal disease.

Association between zinc pool sizes and iron stores in premenopausal women without anaemia.

The simultaneous occurrence of Zn and Fe deficiencies in man has been known since the discovery of human Zn deficiency. However, it is not established that low Fe stores per se or Fe-deficiency anaemia infer low Zn status. Therefore our objective was to identify relationships between Zn and Fe status in premenopausal women without anaemia. We also examined the contribution of food frequencies and blood loss to Zn and Fe status. The subjects were thirty-three apparently healthy premenopausal women without anaemia, who were not taking dietary supplements containing Zn or Fe or oral contraceptives. Main outcomes were Zn kinetic parameters based on the three-compartment mammillary model and serum ferritin (SF) concentration; contributing factors were the frequency of consumption of specific foods and menorrhagia. Lower SF was significantly associated with smaller sizes of Zn pools. The breakpoint in the relationship between SF and the lesser peripheral Zn pool was found to be 21.0 microg SF/l. SF also correlated positively with frequency of beef consumption and negatively with bleeding through menstrual pads (BTMP). Similar to SF, the Zn pool sizes correlated positively with frequency of beef consumption, and negatively with BTMP. In summary, Zn pool sizes and Fe stores were highly correlated in premenopausal women. SF concentrations < 20 microg/l suggest an increased likelihood of low Zn status.

Mercury, lead, and zinc in baby teeth of children with autism versus controls.

This study determined the level of mercury, lead, and zinc in baby teeth of children with autism spectrum disorder (n = 15, age 6.1 +/- 2.2 yr) and typically developing children (n = 11, age = 7 +/- 1.7 yr). Children with autism had significantly (2.1-fold) higher levels of mercury but similar levels of lead and similar levels of zinc. Children with autism also had significantly higher usage of oral antibiotics during their first 12 mo of life, and possibly higher usage of oral antibiotics during their first 36 mo of life. Baby teeth are a good measure of cumulative exposure to toxic metals during fetal development and early infancy, so this study suggests that children with autism had a higher body burden of mercury during fetal/infant development. Antibiotic use is known to almost completely inhibit excretion of mercury in rats due to alteration of gut flora. Thus, higher use of oral antibiotics in the children with autism may have reduced their ability to excrete mercury, and hence may partially explain the higher level in baby teeth. Higher usage of oral antibiotics in infancy may also partially explain the high incidence of chronic gastrointestinal problems in individuals with autism.

Local injection of insulin-zinc stimulates DNA synthesis in skin donor site wound.

Our group has reported the ability of local insulin-zinc injection to accelerate skin wound healing. This experiment was conducted to investigate the metabolic mechanisms of locally injected insulin-zinc in accelerating wound healing. A partial thickness skin donor site wound was created on the back, and indwelling catheters were placed in the carotid artery and jugular vein in anesthetized rabbits. On day 7 after injury, the wound was either injected with 0.2 U of insulin-zinc, 0.4 microg zinc (the amount of zinc in 0.2 U of insulin-zinc), or not injected; stable isotope tracers were infused for measurement of wound DNA synthetic rate as a reflection of cell proliferation. Wound protein synthetic and breakdown rates were also measured. The local insulin-zinc injection increased wound insulin concentration from below 5 pmol/L to 1,260+/-780 pmol/L with minor changes in blood glucose concentration that did not require exogenous glucose replacement. In the insulin-zinc-injected wound, the total DNA synthetic rate was increased by approximately 50% (p<0.05 vs. control). In the zinc-injected wound, whereas total DNA synthetic rate was increased (p<0.05 vs. control), net protein deposition (synthesis-breakdown) was less (p<0.05) than those in the control and insulin-zinc groups. We conclude that local insulin-zinc injection stimulates wound DNA synthesis, which would be expected to accelerate wound reepithelialization.

A 27-amino-acid synthetic peptide corresponding to the NH2-terminal zinc-binding domain of endostatin is responsible for its antitumor activity.

The first recombinant endostatin that elicited strong antitumor activity was expressed in Escherichia coli and administered as a suspension. Under these conditions, the protein retained its full antiangiogenic activity. Lack of requirement for a folded structure prompted us to investigate antitumor properties of synthetic peptides corresponding to different regions of endostatin. Here, we show that the entire antitumor, antimigration, and antipermeability activities of endostatin are mimicked by a 27-amino-acid peptide corresponding to the NH2-terminal domain of endostatin. This peptide contains three histidines that are responsible for zinc binding. Mutations of the zinc-binding histidines abolished its antitumor and antimigration activities, but not antipermeability properties.

Iron release and oxidative DNA damage in splenic toxicity of aniline.

Mechanisms by which aniline produces selective toxicity to the spleen are not well understood. Previously, studies showed that aniline exposure induces lipid peroxidation and protein oxidation in the spleen. The present study was aimed to determine the release of free iron and oxidative DNA damage in the spleen following aniline exposure. To achieve this, male SD rats were orally administered 1 mmol/kg/d aniline for 7 d, while controls received the vehicle only. Total splenic iron content showed a significant increase of 200% in the aniline-treated rats, whereas free iron (low-molecular-weight chelatable iron) showed a marked increase of 375% in comparison to controls. Oxidative DNA damage, measured in terms of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, showed a remarkable increase of 83% in the aniline-treated rats. These results suggest an association between release of free iron and oxidative DNA damage, which could lead to mutagenic and/or carcinogenic responses in the spleen.