Shumao Ding

Intraperitoneal injection of magnetic Fe3O4-nanoparticle induces hepatic and renal tissue injury via oxidative stress in mice

Because of its unique magnetic properties, the iron oxide (Fe3O4) nanoparticle has been widely exploited and its application in various fields has promised immense benefits. However, doubts exist over the use of Fe3O4-nanoparticles in human beings. Thus, the aim of the current study was to find out the potential safety range of medical use. Twenty-five Kunming mice were exposed to Fe3O4-nanoparticles via intraperitoneal injection daily for 1 week at doses of 0, 5, 10, 20, and 40 mg/kg. Hepatic and renal tissues were sliced for physiological observation. Injuries were observed in the high-dose groups (20 and 40 mg/kg) compared with the control group (0 mg/kg). Biomarkers of reactive oxygen species, glutathione, malondialdehyde, DNA-protein crosslinks, and 8-hydroxy-2′-deoxyguanosine in the hepatic and renal tissues were detected. Injury to tissues and oxidative damage to cells at the molecular level was found. The safest dose recommended from the results of this study is 5 mg/kg, as we believe this to be an upper limit balancing the benefits and risks for sub-long-term exposure.

Bone Marrow Injury Induced via Oxidative Stress in Mice by Inhalation Exposure to Formaldehyde

Objective Formaldehyde, a ubiquitous environmental pollutant has been classified as a human leukemogen. However, toxicity of formaldehyde in bone marrow, the target site of leukemia induction, is still poorly understood. Methodology/Principal Findings To investigate bone marrow toxicity (bone marrow pathology, hematotoxicity) and underlying mechanisms (oxidative stress, inflammation, apoptosis) in formaldehyde-exposed mice. Male Balb/c mice were exposed to formaldehyde (0, 0.5, and 3.0 mg/m3) by nose-only inhalation for 8 hours/day, over a two week period designed to simulate a factory work schedule, with an exposure-free “weekend” on days 6 and 7, and were sacrificed on the morning of day 13. Counts of white blood cells, red blood cells and lymphocytes were significantly (p<0.05) decreased at 0.5 mg/m3 (43%, 7%, and 39%, respectively) and 3.0 mg/m3 (52%, 27%, and 43%, respectively) formaldehyde exposure, while platelet counts were significantly increased by 109% (0.5 mg/m3) and 67% (3.0 mg/m3). Biomarkers of oxidative stress (reactive oxygen species, glutathione depletion, cytochrome P450 1A1 and glutathione s-transferase theta 1 expression), inflammation (nuclear factor kappa-B, tomour necrosis factor alpha, interleukin-1 beta), and apoptosis (activity of cysteine-aspartic acid protease 3) in bone marrow tissues were induced at one or both formaldehyde doses mentioned above. Conclusions/Significance Exposure of mice to formaldehyde by inhalation induced bone marrow toxicity, and that oxidative stress, inflammation and the consequential apoptosis jointly constitute potential mechanisms of such induced toxicity.

Inhaled formaldehyde induces DNA-protein crosslinks and oxidative stress in bone marrow and other distant organs of exposed mice.

Formaldehyde (FA), a major industrial chemical and ubiquitous environmental pollutant, has been classified as a leukemogen. The causal relationship remains unclear, however, due to limited evidence that FA induces toxicity in bone marrow, the site of leukemia induction, and in other distal organs. Although induction of DNA–protein crosslinks (DPC), a hallmark of FA toxicity, was not previously detected in the bone marrow of FA‐exposed rats and monkeys in studies published in the 1980s, our recent studies showed increased DPC in the bone marrow, liver, kidney, and testes of exposed Kunming mice. To confirm these preliminary results, in the current study we exposed BALB/c mice to 0, 0.5, 1.0, and 3.0 mg m−3 FA (8 hr per day, for 7 consecutive days) by nose‐only inhalation and measured DPC levels in bone marrow and other organs of exposed mice. As oxidative stress is a potential mechanism of FA toxicity, we also measured glutathione (GSH), reactive oxygen species (ROS), and malondialdehyde (MDA), in the bone marrow, peripheral blood mononuclear cells, lung, liver, spleen, and testes of exposed mice. Significant dose‐dependent increases in DPC, decreases in GSH, and increases in ROS and MDA were observed in all organs examined (except for DPC in lung). Bone marrow was among the organs with the strongest effects for DPC, GSH, and ROS. In conclusion, exposure of mice to FA by inhalation induced genotoxicity and oxidative stress in bone marrow and other organs. These findings strengthen the biological plausibility of FA‐induced leukemogenesis and systemic toxicity. Environ. Mol. Mutagen. 54:705–718, 2013.

Approach to distribution and accumulation of dibutyl phthalate in rats by immunoassay

Dibutyl phthalate (DBP) is mainly taken up by the general population from food intake. To estimate intake of phthalates, determining distribution and accumulation of DBP in biological materials was a critical need. In this work, we set up two novel approaches with a monoclonal antibody specific to DBP to determine the distribution and accumulation of DBP in vivo. The contents of DBP in liver, kidney, stomach and testes were detected by immunofluorescence assays and indirect competitive ELISA. This data give directly evidence that indicates the distribution and accumulation of DBP in vivo. Double-label immunofluorescence assay provides with a visual approach to determination of the distribution and accumulation of DBP. It indicated that DBP accumulated in subcutaneous tissue such as sweat gland, hair follicle. Both of immunofluorescence assay and ELISA can be used to detect the content of DBP in biological materials. Our assays showed that DBP accumulated in viscera being rich in fat, such as liver, kidney and could overcome physiological barriers to penetrate testes. The date suggested that the accumulations of DBP exposed through dermal route were less than that of oral route and most of DBP was metabolized in 2 or 3 days.

An Immunoassay for Dibutyl Phthalate Based on Direct Hapten Linkage to the Polystyrene Surface of Microtiter Plates

Background Dibutyl phthalate (DBP) is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed. Methodology/Principal Findings A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA) employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Compared with conjugate coated format (IC50 = 106 ng/mL), the direct hapten coated format (IC50 = 14.6 ng/mL) improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples. Conclusions/Significance The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed hapten coated icELISA can be used as a convenient quantitative tool for the sensitive and accurate monitoring DBP in water, plastic and cosmetic samples.

Reduction of Endogenous Melatonin Accelerates Cognitive Decline in Mice in a Simulated Occupational Formaldehyde Exposure Environment

Individuals afflicted with occupational formaldehyde (FA) exposure often suffer from abnormal behaviors such as aggression, depression, anxiety, sleep disorders, and in particular, cognitive impairments. Coincidentally, clinical patients with melatonin (MT) deficiency also complain of cognitive problems associated with the above mental disorders. Whether and how FA affects endogenous MT metabolism and induces cognitive decline need to be elucidated. To mimic occupational FA exposure environment, 16 healthy adult male mice were exposed to gaseous FA (3 mg/m3) for 7 consecutive days. Results showed that FA exposure impaired spatial memory associated with hippocampal neuronal death. Biochemical analysis revealed that FA exposure elicited an intensive oxidative stress by reducing systemic glutathione levels, in particular, decreasing brain MT concentrations. Inversely, intraperitoneal injection of MT markedly attenuated FA-induced hippocampal neuronal death, restored brain MT levels, and reversed memory decline. At tissue levels, injection of FA into the hippocampus distinctly reduced brain MT concentrations. Furthermore, at cellular and molecular levels, we found that FA directly inactivated MT in vitro and in vivo. These findings suggest that MT supplementation contributes to the rescue of cognitive decline, and may alleviate mental disorders in the occupational FA-exposed human populations.

In vitro study on cytotoxicity and intracellular formaldehyde concentration changes after exposure to formaldehyde and its derivatives.

HeLa cells were exposed to formaldehyde and its metabolic derivatives, methanol, formic acid, and acetaldehyde, to investigate that the toxicity of formaldehyde is not caused by the chemical group. After 1 h of treatment with formaldehyde, mitochondrial assays showed that low concentrations (e.g. 10 μmol/L) of formaldehyde promoted growth of the HeLa cells, while higher concentrations (e.g. ≥62.5 μmol/L) inhibited cell growth; while all four chemicals at a concentration of 125 μmol/L affected cell growth, formaldehyde affected the largest. Reactive oxygen species concentration increased with the concentration of the exposure chemical. The endogenous formaldehyde content increased the most in the formaldehyde group, but in other three groups, it did not increase as the exposure concentration increased. Expression of dehydrogenase (formaldehyde dehydrogenase (FDH)) in the formaldehyde (10.40) and methanol (10.60) groups increased significantly compared with the control (1), while it was similar to the control in formic acid (0.90) and acetaldehyde (1.10) groups. Our results suggest that formaldehyde could affect cell activity and even enter cells. Exposure to formaldehyde changes the endogenous formaldehyde concentration in cells within 24 h, and this induces expression of FDH for formaldehyde degradation to maintain the formaldehyde balance. The toxicity of formaldehyde is not caused by the carbon atoms in the aldehyde, hydroxyl, or carboxyl groups. Formaldehyde is hypothesized to be an important signaling molecule in the regulation of cell growth and maintenance of the endogenous formaldehyde level.

Formaldehyde-induced paxillin-tyrosine phosphorylation and paxillin and P53 downexpression in Hela cells.

Abstract Formaldehyde (FA) is an environmental pollutant and an endogenous product believed to be involved in tumorigenesis. However, the underlying mechanism of observed FA effects has not been clearly defined. Paxillin is a focal adhesion protein that may play an important role in several signaling pathways. Many paxillin-interacting proteins are involved in the regulation of actin cytoskeleton organization, which is necessary for cell motility events associated with diverse biological responses, such as embryonic development, wound repair and tumor metastasis. P53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is involved in preventing cancer. In this study, we investigated the effects of FA on paxillin–tyrosine phosphorylation and P53 expression in Hela cells by Western blot and immunofluorescence. Western blot analysis revealed that nonlethal concentrations of FA (0.5, 1.0 and 2.0 mM, with the exposure time for 0.5, 1.0 and 2.0 h, respectively) had downregulated paxillin and wild-type p53 genes expression while upregulated paxillin–tyrosine phosphorylation significantly. At the same time, phosphotyrosine at the focal adhesion sites detected by immunofluorescence assay obviously increased in Hela cells incubated with 2.0 mM FA for 2 h. The results suggested that paxillin and p53 genes expression may be involved in FA-related adverse effects and the mechanism may be involved in paxillin–tyrosine phosphorylation.

Cytotoxicity Effects of Nano-Fe3O4 on HeLa Cells

In order to study the cytotoxicity effects of Nano-Fe3O4 Particles on HeLa Cells, 50µg/mL, 100µg/mL and 200µg/mL concentrations of Nano-Fe3O4 were exposed to HeLa cells, twelve hours later&#65292;single cell gel electrophoresis (SCGE) was used to detect HeLa cell DNA molecule damage, the content of the malondialdehyde (MDA) were determined by thiobarbiturie acid reactive substance method and the activity of super oxide dismutase (SOD) was determined by xanthine oxldase method. The results shown that under the 50µg/mL Nano-Fe3O4 exposure, compared with the control group, neither the tail moment nor the tail DNA% increased (P>0.05), the increase content of MDA also not significant (P>0.05), but the reduce activity of SOD is significant (P<0.05); under the 100µg/mL and 200µg/mL Nano-Fe3O4 exposure, compared with the control group, the increase of tail moment and tail DNA% both significant (P<0.05 or P<0.01), the increase content of MDA also significant&#65288;P<0.05 or P<0.01&#65289;&#65292;the reduce activity of SOD is significant (P<0.01). The results suggest that Nano-Fe3O4 could induce the cytotoxicity on HeLa cells.

Data on megakaryocytes in the bone marrow of mice exposed to formaldehyde

Previously, we reported that occupational exposure to formaldehyde (FA) exposure in factory workers reduced platelet counts, http://dx.doi.org/10.1158/1055-9965.EPI-09-0762[1], while exposure in mice increased platelet counts http://dx.doi.org/10.1371/journal.pone.0074974[2]. Bone marrow megakaryocyte (MK) numbers were also increased in exposed mice, as determined qualitatively. The data presented here are from a quantitative evaluation of MK numbers in the bone marrow histopathological slides from the previous FA exposure experiments in mice. Bone marrow slides were prepared using a single 5 μm section of femur from 2 mice randomly selected from each exposure group (n=9) treated with 0, 0.5 and 3.0 mg/m3 FA by nose-only inhalation. MKs were systemically counted and average MK frequency was calculated as the total MK per slide divided by the number of fields evaluated. Data are presented visually as microscopy views and graphically as MK frequency.