Nanometer-long Ge-imogolite nanotubes cause sustained lung inflammation and fibrosis in rats.

BACKGROUND: Ge-imogolites are short aluminogermanate tubular nanomaterials with attractive prospected industrial applications. In view of their nano-scale dimensions and high aspect ratio, they should be examined for their potential to cause respiratory toxicity. Here, we evaluated the respiratory biopersistence and lung toxicity of 2 samples of nanometer-long Ge-imogolites. METHODS: Rats were intra-tracheally instilled with single wall (SW, 70 nm length) or double wall (DW, 62 nm length) Ge-imogolites (0.02-2 mg/rat), as well as with crocidolite and the hard metal particles WC-Co, as positive controls. The biopersistence of Ge-imogolites and their localization in the lung were assessed by ICP-MS, X-ray fluorescence, absorption spectroscopy and computed micro-tomography. Acute inflammation and genotoxicity (micronuclei in isolated type II pneumocytes) was assessed 3 d post-exposure; chronic inflammation and fibrosis after 2 m. RESULTS: Cytotoxic and inflammatory responses were shown in bronchoalveolar lavage 3 d after instillation with Ge-imogolites. Sixty days after exposure, a persistent dose-dependent inflammation was still observed. Total lung collagen, reflected by hydroxyproline lung content, was increased after SW and DW Ge-imogolites. Histology revealed lung fibre reorganization and accumulation in granulomas with epithelioid cells and foamy macrophages and thickening of the alveolar walls. Overall, the inflammatory and fibrotic responses induced by SW and DW Ge-imogolites were more severe (on a mass dose basis) than those induced by crocidolite. A persistent fraction of Ge-imogolites (15% of initial dose) was mostly detected as intact structures in rat lungs 2 m after instillation and was localized in fibrotic alveolar areas. In vivo induction of micronuclei was significantly increased 3 d after SW and DW Ge-imogolite instillation at non-inflammatory doses, indicating the contribution of primary genotoxicity. CONCLUSIONS: We showed that nm-long Ge-imogolites persist in the lung and promote genotoxicity, sustained inflammation and fibrosis, indicating that short high aspect ratio nanomaterials should not be considered as innocuous materials. Our data also suggest that Ge-imogolite structure and external surface determine their toxic activity.

Whole body potassium as a biomarker for potassium uptake using a mouse model.

Potassium is known for its effect on modifiable chronic diseases like hypertension, cardiac disease, diabetes (type-2), and bone health. In this study, a new method, neutron generator based neutron activation analysis (NAA), was utilized to measure potassium (K) in mouse carcasses. A DD110 neutron generator based NAA assembly was used for irradiation.Thirty-two postmortem mice (n= 16 males and 16 females, average weight [Formula: see text] and [Formula: see text] g) were employed for this study. Soft-tissue equivalent mouse phantoms were prepared for the calibration. All mice were irradiated for 10 minutes, and the gamma spectrum with 42K was collected using a high efficiency, high purity germanium (HPGe) detector. A lead shielding assembly was designed and developed around the HPGe detector to obtain an improved detection limit. Each mouse sample was irradiated and measured twice to reduce uncertainty. The average potassium concentration was found to be significantly higher in males [Formula: see text] compared to females [Formula: see text]. We also observed a significant correlation between potassium concentration and the weight of the mice. The detection limit for potassium quantification with the NAA system was 46 ppm. The radiation dose to the mouse was approximately 56 [Formula: see text] mSv for 10-min irradiation. In conclusion, this method is suitable for estimating individual potassium concentration in small animals. The direct evaluation of total body potassium in small animals provides a new way to estimate potassium uptake in animal models. This method can be adapted later to quantify potassium in the human hand and small animals in vivo. When used in vivo, it is also expected to be a valuable tool for longitudinal assessment, kinetics, and health outcomes.

Metabolic fate of ultratrace levels of GeCl(4) in the rat and in vitro studies on its basal cytotoxicity and carcinogenic potential in Balb/3T3 and HaCaT cell linesdagger.

The use of germanium (Ge) and the possibility of exposure to trace and ultratrace amounts of this element is increasing. Germanium is widely used in the industrial field as a semiconductor and also as a dietary supplement, an elixir to 'promote health and cure disease' (e.g. cancer and AIDS). More recently, germanium nanoparticles, ranging in size from 60 to 80 nm, have been developed as a potential spleen imaging agent. Like other metal-based nanoparticles used in nanomedicine, Ge nanoparticles may release trace and ultratrace amounts of Ge ions when injected. The metabolic fate and toxicity of these ions still needs to be evaluated. In this study the metabolic fate of a cationic tetravalent Ge species was studied in vivo by injecting rats i.p. with ultratrace amounts of Ge (80 ng kg(-1)) as [(68)Ge]GeCl(4). The cytotoxicity and carcinogenic potential was assessed in vitro using immortalised human skin keratinocytes and mouse fibroblasts (HaCaT and Balb/c 3T3 cell lines, respectively). At 24 h post-exposure Ge was poorly retained in rat tissues (kidney, liver, intestine, femur, spleen and the heart were the organs with the highest Ge concentration). In the blood, Ge was rapidly cleared, being almost equally distributed between plasma and red blood cells. The excretion was mainly via the urine. The hepatic and renal intracellular distribution showed the highest recovery of Ge in the cytosol and the nuclear fractions. Chromatographic separation and ultrafiltration experiments on kidney and liver cytosols showed that the bulk of Ge was associated with low molecular weight components, representing a 'mobile pool' of the element in the body. However, a significant part of the element was able to interact with biological macromolecules which could be responsible for the presence of Ge in the liver and kidney after 7 days. The in vitro experiments confirmed the low degree of cytotoxicity of GeCl(4) both in HaCaT and Balb/3T3. The latter model was more sensitive to the toxic effects induced by Ge as shown by a colony forming efficiency (CFE) greater than 70% at 700 microm of exposure. At the highest exposure concentration tested (700 microm) GeCl(4) failed to induce morphological neoplastic transformation of the cells, suggesting for the first time that a cationic form of Ge ions has no carcinogenic potential. This supports the results of the only study reported in mice, treated orally long-term to an anionic species of Ge such as sodium germanate (Kanisawa and Schroeder, 1967).

Functional changes on ascending auditory pathway in rats caused by germanium dioxide exposure: an electrophysiological study.

The semiconductor element, germanium (Ge), is essential for the manufacture of modern integrated circuits. Because of its anti-tumor and immunomodulative effects, Ge-containing compounds are also used as health-promoting ingredients in food. However, some histological studies have shown the toxic effects of Ge-containing compounds on various organs, including the central nervous system. Even now, the effect of germanium on auditory system function is not completely clear. To clarify this question, brainstem auditory evoked potentials (BAEPs) were applied to examine the effect of germanium dioxide (GeO(2)) on the ascending auditory pathway. Since the voltage-gated sodium channel is important to neuron activation and nerve conduction, the effect of GeO(2) on voltage-gated sodium channels was also examined. The result revealed GeO(2) elevated the BAEPs threshold dose-dependently. GeO(2) also prolonged latencies and interpeak latencies (IPLs) of BAEPs, but the amplitudes of suprathreshold intensities (90dB) did not show any obvious change. In addition, the results of whole cell patch clamp studies indicated GeO(2) reduced inward sodium current. These results suggest the toxic effect of GeO(2) on the conduction of the auditory system, and that inhibitory effect of GeO(2) on the voltage-gated sodium channels might play a role in GeO(2)-induced abnormal hearing loss.

Role of mitochondrial dysfunction and mitochondrial DNA mutations in age-related hearing loss.

Mitochondrial DNA (mtDNA) mutations/deletions are considered to be associated with the development of age-related hearing loss (AHL). We assessed the role of accumulation of mtDNA mutations in the development of AHL using Polg(D257A) knock-in mouse, which exhibited increased spontaneous mtDNA mutation rates during aging and showed accelerated aging primarily due to increased apoptosis. They exhibited moderate hearing loss and degeneration of the hair cells, spiral ganglion cells and stria vascularis by 9 month of age, while wild-type animals did not. We next examined if mitochondrial damage induced by systemic application of germanium dioxide caused progressive hearing loss and cochlear damage. Guinea pigs and mice given germanium dioxide exhibited degeneration of the muscles and kidney and developed hearing loss due to degeneration of cochlear tissues, including the stria vascularis. Calorie restriction, which causes a metabolic shift toward increased energy metabolism in some organs, has been shown to attenuate AHL and age-related cochlear degeneration and to lower quantity of mtDNA deletions in the cochlea of mammals. Together these findings indicate that decreased energy metabolism due to accumulation of mtDNA mutations/deletions and decline of respiratory chain function play an important role in the manifestation of AHL.

Preparation of germanium apatite and its cytotoxicity.

Germanium apatite was synthesized via the solid-state reaction between GeO(2) and (NH(4))(3)PO(4). The synthesized materials were characterized using XRD, and thermal analysis was carried out using TG-DTA. Ge(2)P(2)O(7) was preferentially produced at temperatures between 300-900 degrees C, and at temperatures above 1000 degrees C, germanium apatite (Ge(5)O(PO(4))(6), GeAp) was synthesized. In solubility tests, 0.36% and 0.65% of Ge ions were liberated from GeAp powder in distilled water at 37 and 80 degrees C after four weeks, respectively. A GeAp aqueous solution maintained at 37 degrees C was strongly acidic with a pH=1.67 after four weeks. The growth rate of human adult gingival fibroblast cells in a medium that included GeAp, HA, and GeO(2) was investigated. The growth rate of the cells in a 0.1 mg/ml GeAp medium was almost the same as that in the control. The cell growth was restricted in a 1.0 mg/ml GeAp medium, whereas the cell growth in a pH-adjusted 1.0 mg/ml GeAp medium at pH=7.60 was higher than that in non-adjusted medium at pH = 7.06.

Potential drugs for elimination of acute lymphatic leukemia cells from autologous bone marrow.

Five drugs, selected because of minimal in vivo myelotoxicity, have been investigated for inhibition of the growth of three acute lymphatic leukemia-derived cell lines. Granulocyte-macrophage colony-forming units (GM-CFU) inhibition with these five drugs after 60-min incubation was first established. Drug concentrations giving up to 90% kill of GM-CFU were then used. Spirogermanium and L-asparaginase did not have an effect on any of the three cell lines under the culture conditions tested, while 4-hydroperoxycyclophosphamide (4-HC) and vincristine inhibited the growth of all three cell lines tested, and bleomycin inhibited the growth of two cell lines. In addition to 4-HC, bleomycin and vincristine should be considered as possible agents in eliminating leukemic cells from autologous marrow grafts. These drugs also deserve further investigation in clonal systems.

Dissolution chemistry and biocompatibility of silicon- and germanium-based semiconductors for transient electronics.

Semiconducting materials are central to the development of high-performance electronics that are capable of dissolving completely when immersed in aqueous solutions, groundwater, or biofluids, for applications in temporary biomedical implants, environmentally degradable sensors, and other systems. The results reported here include comprehensive studies of the dissolution by hydrolysis of polycrystalline silicon, amorphous silicon, silicon-germanium, and germanium in aqueous solutions of various pH values and temperatures. In vitro cellular toxicity evaluations demonstrate the biocompatibility of the materials and end products of dissolution, thereby supporting their potential for use in biodegradable electronics. A fully dissolvable thin-film solar cell illustrates the ability to integrate these semiconductors into functional systems.

Germanium oxide inhibits the transition from G2 to M phase of CHO cells.

We report here for the first time that germanium oxide (GeO(2)) blocks cell progression. GeO(2) is not genotoxic to Chinese hamster ovary (CHO) cells and has limited cytotoxicity. However, GeO(2) arrests cells at G2/M phase. The proportion of cells stopped at G2/M phase increased dose-dependently up to 5 mM GeO(2) when treated for 12 h, but decreased at GeO(2) concentration was greater than 5 mM. Analysis of 5-bromodeoxyuridine-labeled cells indicated that GeO(2) delayed S phase progression in a dose-dependent manner, and blocked cells at G2/M phase. Microscopic examination confirmed that GeO(2) treatment arrested cells at G2 phase. Similar to several other events that cause G2 block, the GeO(2)-induced G2 block can also be ameliorated by caffeine in a dose- and time-dependent manner. To explore the mechanism of G2 arrest by GeO(2), cyclin content and cyclin-dependent kinase activity were examined. Cyclin B1 level was not affected after GeO(2) treatment in CHO cells. However, GeO(2) decreased p34(cdc2) kinase (Cdk1) activity. The kinase activity recovered within 9 h after GeO(2) removal and correlated with the transition of G2/M-G1 phase of the cells. This result suggests that GeO(2) treatment reduces Cdk1 activity and causing the G2 arrest in CHO cells.

Mutagenicity, carcinogenicity and teratogenicity of germanium compounds.

The metalloid germanium has found widespread application in electronics, nuclear sciences and in medicine. General toxicity of germanium is low, except for the tetrahydride germane, and few observations on toxicity of germanium in man exist. Germanium is not carcinogenic and even appears to inhibit cancer development and, in the form of the organic germanium compound, spirogermanium, to destroy cancer cells. Germanium compounds have no mutagenic activity and may, under certain conditions, inhibit the mutagenic activity of other substances. High doses of germanium may result in an increased embryonic resorption, but possible malformations have been reported only after administration of dimethyl germanium oxide to pregnant animals. Germanium may thus be considered an element of rather low risk to man.

Acute and subacute inhalation toxicity of germanium dioxide in rats.

Two acute (4 hr) and one subacute (4 wk) inhalation toxicity studies on germanium dioxide (purity > or = 99%, mean particle size 1.7-2.6 microns) were conducted in young adult Wistar rats. In the acute studies, exposure of two groups of five rats of each sex to maximum attainable concentrations of either 3.10 g amorphous or 1.42 g hexagonal germanium dioxide/m3 for 4 hr was not lethal. In the subacute study, four groups of five rats of each sex were exposed to 0, 16, 72 and 309 mg hexagonal germanium dioxide/m3 for 6 hr/day, 5 days/wk during 4 wk. Two additional groups of 5 rats per sex, exposed either to 0 or to 309 mg/m3, were kept for a 33-day post-exposure period. At the end of the treatment period, changes were observed only in rats of the high concentration group: these changes were decreased body weight gain (both sexes), decreases in haematocrit (females) and thrombocyte count (both sexes), and increases in neutrophil count (both sexes) and white blood cell count (females). On clinical chemistry evaluation, decreased fasting blood glucose (females), decreased total protein concentration (both sexes), increased plasma alanine aminotransferase and aspartate aminotransferase activities (females), increased plasma urea nitrogen (males) and increased plasma bilirubin level (females) were observed. In addition, urinary volume was elevated, and urine density and pH were lowered in both sexes. Relative weights of kidneys, spleen, heart and lungs were higher than in controls. Microscopic examination revealed effects on renal tubular epithelium. Effects on growth, kidneys, and liver were still present at the end of the 33-day recovery period. It was concluded that the 4-hr LC50 value of amorphous germanium dioxide was greater than 3.10 g/m3 and that of the hexagonal form greater than 1.42 g/m3. The no-adverse-effect-level in the 4-wk study using hexagonal germanium dioxide was 72 mg/m3.

Acute and sub-acute inhalation toxicity of germanium metal powder in rats.

An acute (4-hr) and a sub-acute (4-wk) inhalation toxicity study of germanium metal powder (purity 99.8%, mean particle size 2.0-2.4 microns) were carried out in young adult Wistar rats. Exposure of five male and five female rats to 3.86 or 5.34 g/m3 for 4 hr resulted in the death of one rat at each exposure level. Four groups of five male and five female rats were exposed to 0, 9.9, 65.1 or 251.4 mg/m3 for 6 hr/day, 5 days/wk for 30 days. Two additional (recovery) groups of five male and five female rats exposed to 0 or 251.4 mg/m3 were kept untreated for 31 days after exposure. At the end of the treatment period, fasting blood glucose was decreased in males exposed to the high concentration. In females of this group, blood creatinine and urea levels, and urine volumes were increased, but urine density was decreased. Increased blood creatinine levels and urine volume and decreased urine density were also observed in females exposed to 65.1 mg/m3. The absolute and relative lung weights were increased in rats in the mid-and high-concentration groups. Histopathological examination revealed: accumulation of particulate material in the lungs of all treated groups, accumulation of alveolar macrophages in the mid- and high-concentration groups, and alveolitis mainly in the high-concentration group. After the 4-wk recovery period, urine volume was increased in males that had been exposed to germanium. In exposed rats of both sexes, lung weights were still increased and histopathological changes were present, but to a lesser extent than at the end of the exposure period. It was concluded that the 4-hr LC50 value of germanium metal powder in rats is greater than 5.34 g/m3. The no-adverse-effect level in the 4-wk study was 9.9 mg/m3 air.

Therapeutic effects of organic germanium.

Germanium is present in all living plant and animal matter in micro-trace quantities. Its therapeutic attributes include immuno-enhancement, oxygen enrichment, free radical scavenging, analgesia and heavy metal detoxification. Toxicological studies document Germanium's rapid absorption and elimination from the body, and its safety. Clinical trials and use in private practices for more than a decade have demonstrated Germanium's efficacy in treating a wide range of serious afflictions, including cancer, arthritis and senile osteoporosis. Germanium's anti-viral and immunological properties, including the induction of interferon, macrophages, T-suppressor cells and augmentation of natural killer cell activity, suggest its possible efficacy in treating and/or preventing AIDS.

Enzyme histochemical study of germanium dioxide-induced mitochondrial myopathy in rats.

The purpose of this study were 1) to determine the earliest pathological changes of germanium dioxide (GeO2)-induced myopathy; 2) to determine the pathomechanism of GeO2-induced myopathy; and 3) to determine the minimal dose of GeO2 to induce myopathy in rats. One hundred and twenty five male and female Sprague-Dawley rats, each weighing about 150 gm, were divided into seven groups according to daily doses of GeO2. Within each group, histopathological studies were done at 4, 8, 16, and 24 weeks of GeO2 administration. Characteristic mitochondrial myopathy was induced in the groups treated daily with 10 mg/kg of GeO2 or more. In conclusion, the results were as follows: 1) The earliest pathological change on electron microscope was the abnormalities of mitochondrial shape, size and increased number of mitochondria; 2) The earliest pathological change on light microscope was the presence of ragged red fibers which showed enhanced subsarcolemmal succinate dehydrogenase and cytochrome c oxidase reactivity; 3) GeO2 seemed to affect the mitochondrial oxidative metabolism of muscle fibers; 4) GeO2 could induce mitochondrial myopathy with 10 mg/kg of GeO2 for 4 weeks or less duration in rats.

Influence of propagermanium (SK-818) on chemically induced renal lesions in rats.

A histopathological study was performed to examine the influence of propagermanium and germanium dioxide (GeO2) on chemically induced renal lesions in rats. Animals were treated with adriamycin or mercuric chloride to induce glomerular or proximal tubular damage, and then given drinking water containing propagermanium (480 or 2,400 ppm solution) or GeO2 (300 or 1,500 ppm solution: equivalent to propagermanium in terms of germanium contents). The distal tubular epithelium after 8 weeks dosage with the 1,500 ppm solution of GeO2 was characterized by vacuolization and deposits of PAS-positive material not only in adriamycin-treated rats, but also in normal rats. In contrast, propagermanium administration was not associated with any alternation in the changes induced by adriamycin or mercuric chloride. We previously clarified that propagermanium had no biochemical influence on the renal function of these renal injured rats. The histological demonstration that this compound does not exert renal toxicity, even when given at a high dosage to renal injured rats, further indicates that it would not exacerbate renal dysfunction already present. This confirms that propagermanium may be a safe compound for use in individuals with compromised kidneys.

Nephrotoxicity and neurotoxicity in humans from organogermanium compounds and germanium dioxide.

There is no known biological requirement for germanium (Ge), germanates, or any organogermanium compound. Ge deficiency has not been demonstrated in any animal. The estimated average dietary intake of Ge in humans is 1.5 mg/d. Ge is widely distributed in edible foods, all of which, with few exceptions, contain less than 5 ppm Ge, since higher levels are toxic to most plants. Ingestion of Ge compounds has been shown to produce toxic effects in experimental animals. In recent years inorganic germanium salts and novel organogermanium compounds, such as carboxyethyl germanium sesquioxide (Ge-132) and lactate-citrate-germanate (Ge lactate citrate) have been sold as "nutritional supplements" in some countries for their purported immunomodulatory effects or as health-producing elixirs, resulting in intakes of Ge significantly exceeding the estimated average dietary intake. Since 1982, there have been 18 reported cases of acute renal dysfunction or failure, including two deaths, linked to oral intake of Ge elixirs containing germanium dioxide (GeO2) or Ge-132. In these cases, biopsies show vacuolar degeneration in renal tubular epithelial cells, without proteinuria or hematuria, in the absence of glomerular changes. Serum creatinine levels have been well above 400 mumol/L in such patients. In 17 of 18 cases, accumulated elemental Ge intakes reportedly ranged between 16 to 328 g over a 4-36 mo period, or between 100 to 2000 times the average estimated dietary intake for human. In surviving patients, renal function improved after discontinuation of Ge supplementation. However, in no case was recovery complete. One organogermanium compound, an azaspiran organogermanium compound, 2-aza-8-germanspiro[4,5] decane-2-propamine-8,8-diethyl-N,N-dimethyl dichloride (spirogermanium), has been found to cause both neurotoxicity and pulmonary toxicity in phase I and II studies examining its chemotherapeutic potential as an antitumor drug in the treatment of various malignancies. In cancer patients given the drug spirogermanium, 40% experienced marked, yet transient neurotoxicity. Two patients suffered from pulmonary toxicity. Results of phases I and II human cancer trials for spirogermanium have not been favorable, with the exception of moderate benefits for three types of malignancies. It is recommended that patients exposed to long-term (greater than 3 mo) Ge supplementation at levels well above the estimated daily intake be medically supervised and monitored for potential renal-, pulmonary- or neurotoxicity. Further study regarding the mechanism of Ge-induced nephrotoxicity in human is warranted.

[Evaluation of germanium dioxide neurotoxicity in rats and monkeys].

Toxic side effects of germanium dioxide contained in drugs that promote health, include nephropathy, anemia and peripheral neuropathy. Although the neuropathy, which we are interested in, is believed to occur in the patients taking excessive amounts of germanium dioxide, the pathogenesis of such neuropathy is not well understood. Therefore, we studied whether germanium dioxide causes the degeneration of the peripheral nerve in rats and monkeys. Our results showed that in rats, germanium dioxide administered orally and intraperitoneally, 100 mg/kg per day, 3 days a week for 8 weeks and 400 mg/kg per day, once a week for 8 weeks, respectively, did not produce a degeneration of myelinated fibers in teased fiber preparations and Epon-embedded sections of the peripheral nerve. In two monkeys also, germanium dioxide, administered orally, 30 to 40 mg/kg per day, 5 days a week for 8 months, did not produce a degeneration of myelinated fibers of the sural nerve on biopsy, although our results revealed proteinuria and elevated blood urea nitrogen. Further studies are warranted to elucidate the pathogenesis of germanium dioxide induced neuropathy.

Water-soluble germanium nanoparticles cause necrotic cell death and the damage can be attenuated by blocking the transduction of necrotic signaling pathway.

Water-soluble germanium nanoparticles (wsGeNPs) with allyamine-conjugated surfaces were fabricated and emit blue fluorescence under ultraviolet light. The wsGeNP was physically and chemically stable at various experimental conditions. Cytotoxicity of the fabricated wsGeNP was examined. MTT assay demonstrated that wsGeNP possessed high toxicity to cells and clonogenic survival assay further indicated that this effect was not resulted from retarding cell growth. Flow cytometric analysis indicated that wsGeNP did not alter the cell cycle profile but the sub-G1 fraction was absent from treated cells. Results from DNA fragmentation and propidium iodide exclusion assays also suggested that apoptotic cell death did not occur in cells treated with wsGeNP. Addition of a necrosis inhibitor, necrostatin-1, attenuated cell damage and indicated that wsGeNP caused necrotic cell death. Cell signaling leads to necrotic death was investigated. Intracellular calcium and reactive oxygen species (ROS) levels were increased upon wsGeNP treatment. These effects can be abrogated by BAPTA-AM and N-acetyl cysteine respectively, resulting in a reduction in cell damage. In addition, wsGeNP caused a decrease in mitochondrial membrane potential (MMP) which could be recovered by cyclosporine A. The cellular signaling events revealed that wsGeNP increase the cellular calcium level which enhances the production of ROS and leads to a reduction of MMP, consequentially results in necrotic cell death.