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Improved in situ hybridization and G-banding by pretreatment with Denhardt's solution and gelatin-chrome alum.

Various pretreatments of metaphase spreads were examined to obtain optimal DNA labelling patterns while maintaining chromosome integrity during in situ hybridization procedures. Preparations of African green monkey (AGM) chromosomes fixed in methanol-acetic acid (CV-1 cell line) were treated by coating with Denhardt's solution, dilute gelatin-chrome alum, nonfat instant dry milk dissolved in saline-citrate solution (SSC) and/or acetylation prior to denaturation of chromosomal DNA in 70% formamide-2 X SSC for 2 min at 70 degrees C. A 3H-labelled, cloned DNA fragment of the highly repetitive AGM component alpha DNA was hybridized to the chromosomes by incubation at 45 degrees C for 16 h. Treatment with gelatin-chrome alum prior to denaturation greatly improved chromosome morphology and decreased background, but reduced pericentromeric labelling. Sequential treatment with 5 X Denhardt's solution followed by gelatin-chrome alum resulted in enhanced specificity of labelling and excellent chromosome morphology, as well as reduced levels of background. Acetylation had little effect after pretreatment with gelatin-chrome alum, but reduced background levels after pretreatment with Denhardt's solution. Chromosomes treated with Denhardt's solution plus gelatin-chrome alum can be routinely G-banded using trypsin after in situ hybridization.

Animals↗

Chromium deficiency after long-term total parenteral nutrition.

A 63-year-old female developed unexplained hyperglycemia and glycosuria during administration of a total parenteral nutrition regimen on which she had been stable for several months. Because the patient had no history of diabetes or evidence of an infection, chromium deficiency was considered. Plasma chromium level was 0.1 microgram/dl (laboratory reference interval: 1.8-3.8 micrograms/dl). Fourteen days of supplemental intravenous chromium chloride (200 micrograms/day) allowed complete withdrawal of exogenous insulin with no further hyperglycemia or glycosuria. Correction of unexplained glucose intolerance following vigorous chromium supplementation indicates that the patient had chromium deficiency. Subsequent plasma chromium levels remained unchanged, possibly reflecting the sensitivity limits of the assay that was used, the uncertainty that exists regarding appropriate reference intervals for this element, and the fact that plasma levels do not always correlate with total body stores. The patient did not manifest peripheral neuropathy, which was present in one of the two previously reported cases, nor encephalopathy, which was reported in the other. We conclude that this patient developed chromium deficiency as a result of inadequate administration of chromium in the parenteral formula (6 micrograms/day) plus excessive enteric losses, and she presented with glucose intolerance as the only clinical manifestation of the deficiency. Caution should be exercised when interpreting plasma chromium in patients with suspected deficiency.

Blood Glucose↗

Gallocyanin chromalum as a nuclear stain in cytology. I. A cytophotometric comparison of the Husain-Watts Gallocyanin chromalum staining protocol with the Feulgen procedure.

In the present study, the staining characteristics of the Gallocyanin chromalum technique devised by Husain and Watts are compared with the Feulgen reaction. Liver imprints, blood smears, and cervical smears were fixed in ethanol and stained with either the Husain and Watts Gallocyanin chromalum reagent or the Feulgen-Schiff reagent. The slides were then post-treated with 70% ethanol-HCl pH 1.0, or with phosphotungstic acid for 0.5-30 min. The integrated optical density of cell nuclei was measured with a VIDAS image analyzer. In the material stained with the Husain and Watts procedure, some Gallocyanin chromalum was removed from the nuclei in the early phase (5 min) of all the post-treatment steps, followed by a plateau phase where the integrated optical density remained constant for 30 min. In this phase, the nuclear absorbance was highly reproducible and of the same size regardless of the post-treatment. Both the Husain and Watts procedure and the Feulgen-reaction gave quantitative staining of DNA. The Gallocyanin chromalum stain after Husain and Watts is a quick staining procedure for quantitative evaluation of DNA in cytological material. Proper rinsing of the slides is necessary for a good reproducibility of results.

Blood Cells↗

Death by tanning--a case of fatal basic chromium sulphate poisoning.

A woman ingested 400 ml of leather tanning solution containing 48 g of basic chromium sulphate (CrOHSO4). This substance forms hydrogen ions and trivalent chromium when it reacts with tissue proteins. The patient died of cardiogenic shock, complicated by pancreatitis and gut mucosal necrosis and haemorrhage. There are no reported cases of toxicity due to oral ingestion of trivalent chromium. Toxicity of hexavalent and trivalent chromium is discussed and suggestions made for management of future cases.

Adult↗

Isolation and characterization of heavy metals resistant bacteria from Lagos Lagoon.

A total of 228 bacteria with an ability to resist toxic heavy metals were isolated from 8 selected sites of the Lagos Lagoon. The bacteria isolated were Staphylococcus sp., Bacillus sp., Pseudomonas sp., Streptococcus sp., Moraxella sp., Escherichia coli, Proteus sp., Klebsiella sp. and Salmonella sp. The heavy metals to which resistance was recorded were mercury, lead, zinc, cobalt, copper and chromium. The lagoon sites from which the highest number of resistant bacteria were isolated were Marina and Ebute-Ero. The heavy metal to which most bacteria were resistant was cobalt, while the least was chromium. The significance of the result is discussed in relation to the Nigerian environment and human health.

Chlorides↗

Subchronic inhalation toxicity of soluble hexavalent chromium trioxide in rats.

We performed a 90-day repeated-dose inhalation toxicity study of soluble hexavalent chromium trioxide (CrO3 (VI)). Male Sprague-Dawley (SD) rats were exposed to doses of CrO3 in the form of 0.5 approximately 5.0 microm aerosol at 0.00, 0.20, 0.50, and 1.25 mg/m3 for 6 h/day, 5 days/week for 13 weeks using inhalation chamber. CrO3 induced decrease of activity, alopecia and nasal hemorrhage. Body weights of the high-dose 1.25-mg/m3 exposure group were significantly lower than those of the control group. Hematological results revealed the reduction of the number of red blood cell and hematocrit values in the 1.25-mg/m3 exposure group. In addition, the hemoglobin values in the 0.50- and 1.25-mg/m3 exposure groups were significantly decreased compared with those of the control group. Clinical biochemical measurements revealed the reduction in total protein, albumin and alanine aminotransferase (ALT) level of the 0.50- and 1.25-mg/m3 exposure groups. Microscopic examination of the lung showed inflammation reactions caused by Cr exposure. In conclusion, the 13-week repeated exposure with soluble CrO3 demonstrated the injury in SD rats with the no observed adverse effect level (NOAEL) under 0.20 mg/m3.

Administration, Inhalation↗

Digestive constraints on an aquatic carnivore: effects of feeding frequency and prey composition on harbor seals.

We hypothesized that increased feeding frequency in captive harbor seals would increase nutrient loads and thus reduce retention time and the digestive efficiency of natural prey. We measured daily feed intake and excretion during 6 feeding trials and fed herring (49% lipid), pollock (22% lipid) or an equal mix of each diet over 24 months. Animals were accustomed to feeding at either high or low frequency. Body mass and intake did not vary with season. Although mean retention times were similar between diets and feeding frequencies, solute and particulate digesta markers separated at high feeding frequency. Consistent dry matter digestibility resulted in greater gut fill from pollock than from herring. Digestible energy intakes from pollock were approximately 25% greater than from either herring or the mixed diet. Lipid digestibility of herring declined from 90% to 50% when lipid intake exceeded 60 g kg(-0.75) day(-1). Our hypothesis of a trade-off between intake and digestion was not supported for protein but was supported for lipid. Results of this study imply that a flexible digestive system for harbor seals can compensate for ingesting prey of lower energy density by increasing gut fill and enhancing protein and lipid assimilation, to sustain digestible energy intake.

Animal Feed↗

Ultrastructural damage in chromium picolinate-treated cells: a TEM study. Transmission electron microscopy.

Chromium picolinate (CrPic) is a human dietary supplement that provides a bioavailable form of chromium(III). Its mechanism of action is unknown, and a number of toxic endpoints have been attributed to its use. Understanding the cellular effects of CrPic is important for confirmation or dismissal of these potential toxic effects. The purpose of this work was to characterize morphological damage caused by CrPic, picolinic acid, and chromic chloride in Chinese hamster ovary AA8 cells. A 48-h exposure to 80 micro g/cm(2) CrPic (0.44 mg/mL CrPic) produced 45% survival by colony formation. Transmission electron microscopy (TEM) showed 83% of analyzed cells having swollen mitochondria with degraded cristae. Apoptosis was identified by nuclear convolution and fragmentation, and cytoplasmic blebbing. Apoptosis was quantified by fluorescence microscopy with acridine orange/ethidium bromide staining. At the 80 micro g/cm(2) dose of CrPic, 37% of the cells were apoptotic cells at 48 h. An equivalent dose of picolinate, 3 mM, was much more cytotoxic and thus there was an inadequate cell number for TEM analysis. However, a lower dose of 1.5 mM induced 49% cell survival, and damaged 86% of the mitochondria, with 51% of the cells undergoing apoptosis. A dose of 1 mM chromic chloride produced 71% cell survival, and damaged 86% of the mitochondria, with 22% of the cells undergoing apoptosis. The amount of apoptosis correlated with overall cell survival by colony formation, but not with the amount of mitochondrial damage. The coordination of Cr(III) by picolinate ligands may alter the cellular chemistry of Cr(III) to make chromium picolinate a toxic form of Cr(III).

Animals↗

Oral administration of the biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ increases insulin sensitivity and improves blood plasma variables in healthy and type 2 diabetic rats.

The in vivo effects of gavage administration of the synthetic, functional biomimetic cation [Cr(3)O(O(2)CCH(2)CH(3))(6)(H(2)O)(3)](+) to healthy and type 2 diabetic model rats are described. After 24 weeks of treatment (0-1,000 microg Cr/kg body mass) of healthy Sprague Dawley rats, the cation results in a lowering (P < 0.05) of fasting blood plasma low-density lipoprotein (LDL) cholesterol, total cholesterol, triglycerides, and insulin levels and of 2-h plasma insulin and glucose concentrations after a glucose challenge. Zucker obese rats (a model of the early stages of type 2 diabetes) and Zucker diabetic fatty rats (a model for type 2 diabetes) after supplementation (1,000 microg Cr/kg) have lower fasting plasma total, high-density lipoprotein, and LDL cholesterol, triglycerides, glycated hemoglobin, and insulin levels and lower 2-h plasma insulin levels in glucose tolerance tests. The lowering of plasma insulin concentrations with little effect on glucose concentrations suggests that the supplement increases insulin sensitivity. The cation after 12 and 22 or 24 weeks of administration lowers (P < 0.05) fasting plasma glycated hemoglobin levels in the Zucker diabetic and Zucker obese rats, respectively, and thus can improve the glucose status of the diabetic models. The effects cannot be attributed to the propionate ligand.

Animals↗

Bone apposition to titanium implants biocoated with recombinant human bone morphogenetic protein-2 (rhBMP-2). A pilot study in dogs.

The aim of the present study was to investigate bone formation to recombinant human bone morphogenetic protein-2 (rhBMP-2)-biocoated and rhBMP-2-nonbiocoated titanium implants after implantation in dogs. Implantation of sand-blasted and acid-etched (C), chromosulfuric acid surface-enhanced (CSA), and rhBMP-2-biocoated CSA [BMP-A: noncovalently immobilized rhBMP-2 (596 ng/cm(2)), BMP-B: covalently immobilized rhBMP-2 (819 ng/cm(2))] implants was performed in both the mandible and tibia of dogs. After 4 weeks of healing, the percentage of direct bone to implant contact (BIC) and the induced bone density (BD) at a distance of less than and greater than 1 mm adjacent to each implant was assessed. Histomorphometric analysis of implants inserted in the mandible and tibia revealed that BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C. BD as measured at a distance of <1 mm revealed obvious differences between groups: BMP-B>BMP-A>CSA>C. However, no differences between groups were observed at a distance of >1 mm. Within the limits of the present study, it may be concluded that rhBMP-2 immobilized by covalent and noncovalent methods on CSA-treated implant surfaces seemed to be stable and promoted direct bone apposition in a concentration-dependent manner.

Animals↗

Bacterial Cr(VI) reduction concurrently improves sunflower (Helianthus Annuus L.) growth.

Four Cr(VI)-reducing bacterial strains (Ochrobactrum intermedium, CrT-2, CrT-3 and CrT-4) previously isolated from chromium-contaminated sites were inoculated on to seeds of sunflower (Helianthus annuus var SF-187), which were germinated and grown along with non-inoculated controls with chromate salts (300 microg CrCl3 or K2CrO4 ml(-1)). Severe reduction (20%) in seed germination was observed in Cr(VI) stress. Plant height decreased (36%) with Cr(VI) when compared with chromium-free control, while O. intermedium inoculation resulted a 20% increment in this parameter as compared to non-inoculated chromium-free control. CrT-3 inoculation resulted a 69% increment in auxin content as compared to non-inoculated control. O. intermedium caused 30% decrease in chromium uptake in sunflower plant roots under Cr(VI) stress as compared to chromium-free control plants.

Biodegradation, Environmental↗