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Titanium is the only metal to which osteoblasts can adhere and on which they can grow and form bone tissue in vivo, resulting in a strong bond between the implant and living bone. This discovery provides the basis for the universal medical application of Ti. However, the biochemical mechanism of bond formation is still unknown. We aimed to elucidate the mechanism of bond formation between collagen, which constitutes the main organic component of bone, and TiO2, of which the entire surface of pure Ti is composed. We analysed the binding between the soluble collagen and TiO2 by chromatography with a column packed with Ti beads of 45 µm, and we explored the association between collagen fibrils and TiO2 (anatase) powders of 0.2 µm. We ran the column of chromatography under various elution conditions. We demonstrated that there is a unique binding affinity between Ti and collagen. This binding capacity was not changed even in the presence of the dissociative solvent 2M urea, but it decreased after heat denaturation of collagen, suggesting the contribution of the triple-helical structure. We propose a possible role of periodically occurring polar amino acids and the collagen molecules in the binding with TiO2., Graphical Abstract

Exposure to cadmium (Cd) is a risk factor to health impairments, wherein its cytotoxicity is attributed to induction of oxidative stress. Usage of anti-oxidants, however, can help lessen the damaging effects of Cd. The effect of Cd interaction with low concentration of dietary anti-oxidants, L-ascorbic acid and (-)-epigallocatechin gallate (EGCG), to PC12 cellular mechanisms was examined. The expected toxicity of Cd was observed on PC12 cells but addition of L-ascorbic acid ameliorated this effect. On the other hand, addition of EGCG was able to increase the cytotoxicity of Cd and to decrease the protective effect of L-ascorbic acid against Cd. Increase in LDH activity and decrease in free sulfhydryl levels indicated cell membrane damage and oxidative stress, respectively, in Cd- and EGCG-Cd-treated cells. Downregulation of pro-apoptotic proteins (pro-caspase-9, p53, and ERK1) was observed in cells treated with Cd alone and EGCG-Cd, while upregulation of autophagy-linked proteins (p62 and pBeclin1) was found on L-ascorbic acid-Cd combination treatments. These findings indicate that Cd causes cells to undergo an autophagy-enhanced cell death; low-concentration EGCG and L-ascorbic acid promotes cell survival individually; however, interaction of EGCG with Cd showed enhancement of Cd toxicity and antagonism of L-ascorbic acid efficiency.

Arsenic is a well-recognized environmental contaminant that occurs naturally through geogenic processes in the aquifer. More than 200 million people around the world are potentially exposed to the elevated level of arsenic mostly from Asia and Latin America. Many adverse health effects including skin diseases (i.e., arsenicosis, hyperkeratosis, pigmentation changes), carcinogenesis, and neurological diseases have been reported due to arsenic exposure. In addition, arsenic has recently been shown to contribute to the onset of non-communicable diseases, such as diabetes mellitus and cardiovascular diseases. The mechanisms involved in arsenic-induced diabetes are pancreatic β-cell dysfunction and death, impaired insulin secretion, insulin resistance and reduced cellular glucose transport. Whereas, the most proposed mechanisms of arsenic-induced hypertension are oxidative stress, disruption of nitric oxide signaling, altered vascular response to neurotransmitters and impaired vascular muscle calcium (Ca2+) signaling, damage of renal, and interference with the renin-angiotensin system (RAS). However, the contributions of arsenic exposure to non-communicable diseases are complex and multifaceted, and little information is available about the molecular mechanisms involved in arsenic-induced non-communicable diseases and also no suitable therapeutic target identified yet. Therefore, in the future, more basic research is necessary to identify the appropriate therapeutic target for the treatment and management of arsenic-induced non-communicable diseases. Several reports demonstrated that a daily balanced diet with proper nutrient supplements (vitamins, micronutrients, natural antioxidants) has shown effective to reduce the damages caused by arsenic exposure. Arsenic detoxication through natural compounds or nutraceuticals is considered a cost-effective treatment/management and researchers should focus on these alternative options. This review paper explores the scenarios of arsenic contamination in groundwater with an emphasis on public health concerns. It also demonstrated arsenic sources, biogeochemistry, toxicity mechanisms with therapeutic targets, arsenic exposure-related human diseases, and onsets of cardiovascular diseases as well as feasible management options for arsenic toxicity.

Mercury (Hg) is a hazardous metal, poses environmental problems with severe human health effects; whereas zinc (Zn) is an essential micronutrient with antioxidant properties. The purpose of this research was to investigate the effect of Zn on inorganic Hg-induced cytotoxicity in the PC12 cells. The cells were treated with HgCl2 (5 μM) for 48 h with/without 1 h prior ZnCl2-treatment (100 μM) and deliberated for further analysis. After 48 h of incubation with only Hg2+, the cell showed reduced cell viability, compromised cell membrane, DNA degradation, depleted glutathione level, ROS generation and drastically increased apoptosis. Subsequently, Hg2+-treated cells demonstrated a significant downregulation of akt, mTOR, ERK1, Nrf2, HO1, Bcl-2, Bcl-xL, and upregulation of p53, Bax, cytochrome c and cleaved caspase 3, indicating intrinsic apoptosis induction. However, cells pretreated with Zn2+ before Hg2+-exposure showed a significant improvement in cell viability, cell membrane, DNA damage, glutathione level, ROS amount and apoptotic cells, with a significant upregulation in mTOR, akt, ERK1, Nrf2, HO1, Bcl-2 and Bcl-xL, and downregulation in p53, Bax, cytochrome c and cleaved caspase 3, indicating inhibition of apoptosis. The findings suggested that Zn2+-pretreatment not only improves glutathione content but also induces activation of Nrf2-HO1 pathway, which would tend to suppress Hg-cytotoxicity.

Green synthesized silver nanoparticles (Ag-NPs) have demonstrated promising effects, including cytotoxicity and anticancer potential, in different cell lines. Therefore, in our previous study, Ag-NPs were synthesized from the reduction of AgNO3 using Brassica rapa var. japonica (Bj) leaf extract as a reducing and stabilizing agent. The synthesized Ag-NPs were spherical in shape, with a size range of 15-30 nm. They had phase-centered cubic structure with strong growth inhibition potential against some bacteria. In continuation with our previous study, in the present study, we aimed to investigate the autophagy-regulated cytotoxic effect of Ag-NPs against human epithelial colorectal adenocarcinoma cells (Caco-2 cells). We found that the Bj leaf aqueous extract facilitated Brassica silver nanoparticles (Brassica Ag-NPs)-induced NF-κB mediated autophagy in Caco-2 cells. Results showed that Ag-NPs reduced cell viability of Caco-2 cells by inducing oxidative stress and DNA damage. Therefore, to understand the mechanism underlying the death-promoting activity of Ag-NPs in Caco-2 cells, western blotting was performed. Western blot analysis showed decreased expression of NFκB and increased expression of IκB, which is a sign of autophagy initiation. In addition, autophagosome formation was accelerated by the activity of p53 and light chain 3 (LC3) II. In addition, inhibition of Akt and mTOR also played a pivotal role in autophagy formation. Finally, excessive expansion of autophagy promoted apoptosis, which subsequently resulted in necrosis. These findings support a novel cell death-promoting function of autophagy by Ag-NPs in Caco-2 cells.

Arsenic is a recognized highly toxic contaminant, responsible for numerous human diseases and affecting many millions of people in different parts of the world. Contrarily, curcumin is a natural dietary polyphenolic compound and the main active ingredient in turmeric. Recently it has drawn great attention due to its diverse biological activities, strong antioxidant properties and therapeutic potential against many human ailments. In this study, we aimed to explore the protective effects and the regulatory role of curcumin on arsenic-induced toxicity and gain insights into biomolecular mechanism/s. Arsenic (10 μM) treatment in PC12 cells for 24 h induced cytotoxicity by decreasing cell viability and intracellular glutathione level and increasing lactate dehydrogenase activity and DNA fragmentation. In addition, arsenic caused apoptotic cell death in PC12 cells, which were confirmed from flow cytometry results. Moreover, arsenic (10 μM) treatment significantly down-regulated the inhibition factors of autophagy/apoptosis; mTOR, Akt, Nrf2, ERK1, Bcl-x, Xiap protein expressions, up-regulated the enhanced factors of autophagy/apoptosis; ULK, LC3, p53, Bax, cytochrome c, caspase 9, cleaved caspase 3 proteins and eventually caused autophagic and apoptotic cell death. However, curcumin (2.5 μM) pretreatment with arsenic (10 μM) effectively saves PC12 cells against arsenic-induced cytotoxicity through increasing cell viability, intracellular GSH level and boosting the antioxidant defense system, and limiting the LDH activity and DNA damage. Furthermore, pretreatment of curcumin with arsenic expressively alleviated arsenic-induced toxicity and cell death by reversing the expressions of proteins; mTOR, Akt, Nrf2, ERK1, Bcl-x, Xiap, ULK, LC3, p53, Bax, cytochrome c, caspase 9 and cleaved caspase 3. Our findings indicated that curcumin showed antioxidant properties through the Nrf2 antioxidant signaling pathway and alleviates arsenic-triggered toxicity in PC12 cells by regulating autophagy/apoptosis.

Background Previously we found that a group of phosphorylated proteins (SIBLINGs) in bone binds with the Ti-device, and increases the early bone formation around the Ti-implants remarkably. From these results, we explained the biochemical mechanism of a strong bond between living bone and Ti, which was discovered by Branemark and colleagues. For the clinical application of our findings, we need a large amount of these proteins or their substitutes. Objective We aimed to create a new molecule that equips with essential functions of SIBLINGs, Ti-binding, and bone enhancement around the Ti implant. Methods We chemically phosphorylated chitin and obtained a soluble form of phosphorylated chitin (P-chitin). In this solution, we immersed the Ti-devices of web-form (TW) which we previously developed and obtained the P-chitin coated TWs. Then we tested the P-chitin coated TWs for their calcification ability in vitro, and bone enhancing ability in vivo, by implanting them into rat calvaria. We compared the P-chitin coated TW and the non-coated TW in regard to their calcification and bone enhancing abilities. Results Ti-devices coated with phosphorylated-chitin induced a ten times higher calcification in vitro at 20 days, and four times more elevated amount of bone formation in vivo at two weeks than the uncoated Ti-device. Conclusions Phosphorylated chitin could be a partial substitute of bone SIBLING proteins and are clinically applicable to accelerate bone formation around the Ti implants, thereby achieving the strong bond between living bone and Ti.

Arsenic is well known toxicant responsible for human diseases including cancers. On the other hand, selenium is an essential trace element with significant chemopreventive effects, anticancer potentials and antioxidant properties. Although previous studies have reported antagonism/synergism between arsenic and selenium in biological systems, the biomolecular mechanism/s is still inconclusive. Therefore, to elucidate the molecular phenomena in cellular level, we hypothesized that co-exposure of selenium with arsenic may have suppressive effects on arsenic-induced cytotoxicity. We found that selenium in co-exposure with arsenic increases cell viability, and suppresses oxidative stress induced by arsenic in PC12 cells. Consequently, DNA fragmentation due to arsenic exposure was also reduced by arsenic and selenium co-exposure. Furthermore, western blot analyses revealed that simultaneous exposure of both metals significantly inhibited autophagy which further suppressed apoptosis through positively regulation of key proteins; p-mTOR, p-Akt, p-Foxo1A, p62, and expression of ubiquitin, Bax, Bcl2, NFкB, and caspases 3 and 9, although those are negatively regulated by arsenic. In addition, reverse transcriptase PCR analysis confirmed the involvement of caspase cascade in cell death process induced by arsenic and subsequent inhibition by co-exposure of selenium with arsenic. The cellular accumulation study of arsenic in presence/absence of selenium via inductively coupled plasma mass spectrometry confirmed that selenium effectively retarded the uptake of arsenic in PC12 cells. Finally, these findings imply that selenium is capable to modulate arsenic-induced intrinsic apoptosis pathway via enhancement of mTOR/Akt autophagy signaling pathway through employing antioxidant potentials and through inhibiting the cellular accumulation of arsenic in PC12 cells.

The study was designed to collect water samples over two seasons—wet-monsoon season (n = 96) (March–April) and dry-monsoon season (n = 44) (September–October)—to understand the seasonal variation in anion and cation hydrochemistry of the coastal rivers and estuaries contributing in the spatial trend in salinity. Hydrochemical examination of wet-monsoon season primarily revealed Ca–Mg–HCO3 type (66%) and followed by Na–Cl type (17.70%) water. In the dry-monsoon season, the scenario reversed with primary water being Na–Cl type (52.27%) followed by Ca–Mg–HCO3 type (31.81%). Analysis of Cl/Br molar ratio vs. Cl (mg/L) depicted sampling area affected by seawater intrusion (SWI). Spatial analysis by ordinary kriging method confirmed approximately 77% sample in the dry-monsoon, and 34% of the wet-monsoon season had shown SWI. The most saline-intruded areas in the wet-monsoon seasons were extreme south-west coastal zone of Bangladesh, lower Meghna River floodplain and Meghna estuarine floodplain and south-eastern part of Chittagong coastal plains containing the districts of Chittagong and Cox’s Bazar adjacent to Bay of Bengal. In addition, mid-south zone is also affected slightly in the dry-monsoon season. From the analyses of data, this study could further help to comprehend seasonal trends in the hydrochemistry and water quality of the coastal and estuarine rivers. In addition, it can help policy makers to obligate some important implications for the future initiatives taken for the management of land, water, fishery, agriculture and environment of coastal rivers and estuaries of Bangladesh.

Mercury (Hg) in its all forms, including inorganic Hg (iHg) is an environmental contaminant due to toxicity and diseases in human. However, a little is known about the underlying mechanisms responsible for iHg toxicity. Selenium (Se) is an essential trace element, recognized as an antioxidant and protective agent against metal toxicities. The purpose of this research was to investigate ameliorations of Se counter to iHg-mediated toxicity in PC12 cells. Cytotoxic assays have been shown that iHg (5 μM) caused oxidative stress and intrinsic apoptosis via ROS generation, oxidizing glutathione, damaging DNA, degrading cell membrane integrity, down-regulating mTOR, p-mTOR, akt and ERK1, and up-regulating cleaved caspase 3 and cytochrome c release in PC12 cells 48 h after incubation. Co-treatment of Se (5 μM) inhibited intrinsic apoptosis and oxidative stress induced by iHg (5 μM) via inhibiting ROS formation, boosting GPx contents, increasing reduced glutathione, limiting DNA degradation, improving cell membrane integrity, up-regulating mTOR, p-mTOR, akt, ERK1 and caspase 3, and down-regulating cleaved caspase 3 and cytochrome c leakage in PC12 cells. In conclusion, these results recommended that excessive ROS generation acts a critical role in iHg-influenced oxidative stress and co-treatment of Se attenuates iHg-cytotoxicity through its antioxidant properties.

Mercury (Hg) is a toxic metal, well-known for its dangerous health effects on human. Butylated hydroxytoluene (BHT) is a phenolic component generally consumed as a food additive as an antioxidant. However, BHT induced antioxidant properties against heavy metals-influenced toxicity are little studied. We hypothesized that BHT has a regulatory effect on Hg-induced cytotoxicity. The objective of this research was to assess the protecting effects of BHT against inorganic Hg (iHg)-toxicity in PC12 cells, where cells were treated with/without HgCl2 (Hg2+) (5 μM) and BHT (100 μM) for 48 h and analyzed further. Cells treated by Hg caused a significant cell viability reduction, membrane damage, glutathione reduction, DNA fragmentation, ROS generation, with suppressed expressions of akt, mTOR, ERK1, Nrf2 and HO1; and elevated apoptotic expressions of p53, Bax, cytochrome c and active caspase 3. However, BHT and Hg2+ co-exposure showed prevention against Hg2+-toxicity by improving GSH content and inhibiting ROS generation and oxidative stress mediated damages. Additionally, BHT co-treatment inverted the pro-apoptotic proteins by augmenting pro-survival regulatory proteins akt, mTOR, ERK1, Nrf2 and HO1. These findings proved that BHT inhibits Hg2+-toxicity, hindering ROS generation and intrinsic apoptosis, via enhancing glutathione and antioxidants; and suggested BHT implications as therapeutic.

Mercury (Hg) is an extremely dangerous environmental contaminant, responsible for human diseases including neurological disorders. However, the mechanisms of inorganic Hg (iHg)-induced cell death and toxicity are little known. Dihydrolipoic acid (DHLA) is the reduced form of a naturally occurring compound lipoic acid, which act as a potent antioxidant through multiple mechanisms. So we hypothesized that DHLA has an inhibitory role on iHg-cytotoxicity. The purposes of this research were to investigate mechanism/s of cytotoxicity of iHg, as well as, the cyto-protection of DHLA against iHg induced toxicity using PC12 cells. Treatment of PC12 cells with HgCl2 (Hg2+) (0–2.5 μM) for 48 h resulted in significant toxic effects, such as, cell viability loss, high level of lactate dehydrogenase (LDH) release, DNA damage, cellular glutathione (GSH) level decrease and increased Hg accumulation. In addition, protein level expressions of akt, p-akt, mTOR, GR, NFkB, ERK1, Nrf2 and HO-1 in cells were downregulated; and cleaved caspase 3 and cytochrome c release were upregulated after Hg2+ (2.5 μM) exposure and thus inducing apoptosis. Hg2+induced apoptosis was also confirmed by flow cytometry. However, pretreatment with DHLA (50 μM) for 3 h before Hg2+ (2.5 μM) exposure showed inhibition against iHg2+-induced cytotoxicity by reversing cell viability loss, LDH release, DNA damage, GSH decrease and inhibiting Hg accumulation. Moreover, DHLA pretreatment reversed the protein level expressions of akt, p-akt, mTOR, GR, NFkB, ERK1, Nrf2, HO-1, cleaved caspase 3 and cytochrome c. In conclusion, results showed that DHLA could attenuate Hg2+-induced cytotoxicity via limiting Hg accumulation, boosting up of antioxidant defense, and inhibition of apoptosis in cells.

Arsenic is awfully toxic metalloid responsible for many human diseases all over the world. Contrastingly, D-pinitol is a naturally occurring bioactive dietary compound has antioxidant properties. The objective of this study is to elucidate the protective actions of D-pinitol on arsenic-induced cytotoxicity and explore its controlling role in biomolecular mechanisms in PC12 cells. Obtained results demonstrated that co-exposure of D-pinitol with arsenic increases cell viability, decreases DNA damage and protects PC12 cells from arsenic-induced cytotoxicity by increasing glutathione (GSH) level and glutathione reductase (GR). Protein expression of western blot analysis showed that co-exposure of D-pinitol and arsenic significantly inhibited arsenic-induced autophagy which further suppressed apoptosis through up-regulation of survival factors; mTOR, p-mTOR, Akt, p-Akt, NF-кB, Nrf2, ERK1, GR, Bcl-x and down-regulation of death factors; p53, Bax, cytochrome c, LC3, although arsenic regulated those factors negatively. These results of this study suggested that D-pinitol protects PC12 cells from arsenic-induced cytotoxicity.

Metals are ubiquitous in the environment due to huge industrial applications in the form of different chemicals and from extensive mining activities. The frequent exposures to metals and metalloids are crucial for the human health. Trace metals are beneficial for health whereas non-essential metals are dangerous for the health and some are proven etiological factors for diseases including cancers and neurological disorders. The interactions of essential trace metals such as selenium (Se) and zinc (Zn) with non-essential metals viz. lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) in biological system are very critical and complex. A huge number of studies report the protective role of Se and Zn against metal toxicity, both in animal and cellular levels, and also explain the numerous mechanisms involved. However, it has been considered that a tiny dyshomeostasis in the metals/trace metals status in biological system could induce severe deleterious effects that can manifest to numerous diseases. Thus, in this particular review, we have demonstrated the critical protection mechanism/s of Se and Zn against Cd, Pb, As and Hg toxicity in a one by one manner to clarify the up-to-date findings and perspectives. Furthermore, biomolecular consequences are comprehensively presented in light of particular cellular/biomolecular events which are somehow linked to a subsequent disease. The analyzed reports support significant protection potential of Se and Zn, either alone or in combination with other agents, against each of the abovementioned non-essential metals. However, Se and Zn are still not being used as detoxifying agents due to some unexplained reasons. We hypothesized that Se could be a potential candidate for detoxifying As and Hg regardless of their chemical speciations, but requires intensive clinical trials. However, particularly Zn-Hg interaction warrants more investigations both in animal and cellular level.

Nonylphenol and short-chain nonylphenol ethoxylates such as NP2 EO are present in aquatic environment as wastewater contaminants, and their toxic effects on aquatic species have been reported. Apoptosis has been shown to be induced by serum deprivation or copper treatment. To understand the toxicity of nonylphenol diethoxylate, we investigated the effects of NP2 EO on apoptosis induced by serum deprivation and copper by using PC12 cell system. Nonylphenol diethoxylate itself showed no toxicity and recovered cell viability from apoptosis. In addition, nonylphenol diethoxylate decreased DNA fragmentation caused by apoptosis in PC12 cells. This phenomenon was confirmed after treating apoptotic PC12 cells with nonylphenol diethoxylate, whereas the cytochrome c release into the cytosol decreased as compared to that in apoptotic cells not treated with nonylphenol diethoxylates. Furthermore, Bax contents in apoptotic cells were reduced after exposure to nonylphenol diethoxylate. Thus, nonylphenol diethoxylate has the opposite effect on apoptosis in PC12 cells compared to nonylphenol, which enhances apoptosis induced by serum deprivation. The difference in structure of the two compounds is hypothesized to be responsible for this phenomenon. These results indicated that nonylphenol diethoxylate has capability to affect cell differentiation and development and has potentially harmful effect on organisms because of its unexpected impact on apoptosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1389-1398, 2016.

Arsenic (As) and cadmium (Cd) are noxious and carcinogenic pollutants that can be removed from water by using emerging, ecofriendly, phytofiltration technology that employs Micranthemum umbrosum. After culturing M. umbrosum for 7 days in a hydroponic experiment, accumulation of 1219±44.11 µg As g−1 and 799.40±30.95 µg Cd g−1 were observed in the leaves, from 1000 µg As L−1 and 1000 µg Cd L−1 of water, respectively. Plant and water samples were analyzed for assessing the As and Cd accumulations, translocations, phytotoxic effects, uptake mechanisms and kinetics, and for evaluating the potential of M. umbrosum in As and Cd phytofiltration. The uptake pattern was leaf>stem>root for both pollutants. The plant showed higher resistance to As than to that to Cd. Uptake of inorganic As species was much greater than that of organic As and was found at above the substrate concentration. However, Cd showed similar uptake pattern to that of inorganic As species, and the data was better fit to a non-linear than a linear model. Low molecular weight substances that have thiol group(s) may be responsible for the binding of As in plants whereas Cd showed a different mechanism to that of As. M. umbrosum showed good As phytofiltration capabilities without any phytotoxic effects, but it was found to be a moderate accumulator of Cd with some phytotoxic effect compare to some other previously studied plant.

Purpose of this study is to investigate mechanism/s of cyto-protection by selenium (Na2SeO3; Se4+) against cadmium (CdCl2; Cd2+)-induced cytotoxicity using PC12 cells. In addition, Se (5, 10, 20 and 40 μM) and Cd (2.5, 5 and 10 μM)-induced cytotoxicity is determined. Cytotoxicity assays and western blot analyses confirmed that Se (≥10 μM) promotes autophagic cell death via inhibition of mTOR activation and p62 accumulation due to increase of cellular oxidative stress. On the other hand, co-presence of non-toxic Se (5 μM) and toxic Cd (5 μM) showed to increase cell viability, glutathione and glutathione peroxidase 1 (GPx1) levels, and to decrease DNA fragmentation and lactate dehydrogenase (LDH) activity compared to Cd-treated (5 μM) cells alone. Furthermore, western blot analyses of cytochrome c and ERK1 indicated that Cd-induced apoptotic cell death in PC12 cells. However, the co-exposure of Se with Cd significantly decreases the release of cytochrome c into cytosol from mitochondria, and up-regulates ERK1 protein to inhibit Cd-induced apoptosis. In conclusion, Se (≥10 μM) possess cytotoxicity in PC12 cells; however, co-presence of Se (5 μM) with Cd (5 μM) protects against Cd-induced apoptosis in PC12 cells due to inhibition of Cd-induced oxidative stress and subsequently suppression of mitochondrial apoptosis pathway.

Polyphenols have many beneficial effects and an effective disease therapeutic auxiliary drug. Previously, myricetin, a polyphenol, had been reported to possess various biological effects on human physiology. However, mechanism of myricetin on apoptosis induced in PC12 cells is still unclear. PC12 cells were treated with myricetin in two concentration levels comprising 0.1 and 1 μM under serum-free condition. As a result, morphological changes were observed using trypan blue assay. DNA fragmentation was determined by DNA ladder assay to evaluate DNA damage levels. Western blotting results showed that cytosolic cytochrome c which was released from mitochondria. Subsequently, tumor suppressor gene p53, pro-apoptotic and anti-apoptotic Bcl-2 family proteins Bax and Bcl-2 were expressed. The caspase cascade reaction was induced through caspase 3 and 9 expression. From these results, it is suggested that myricetin significantly enhanced the apoptosis induced by serum deprivation in a dose-dependent manner in PC12 cells.

Despite extensive research on copper toxicity the mechanisms involved are not fully characterized. There have been many recent reports concerning the relationship between epigenetic factors and cell metabolism, but the effects of copper exposure on epigenetic factors have not been investigated. In this study, an in vitro culture system was employed to study the influence of copper on apoptosis and epigenetic factors in PC12 cells. When PC12 cells were exposed to copper, DNA damage was observed as DNA fragmentation. In addition, cytosolic cytochrome c levels were increased by copper treatment. These results suggested that copper induced apoptosis via an oxidative stress pathway. This was consistent with the observation that copper-induced apoptosis was enhanced by further oxidative stress induced by exposing cells to H₂O₂. In addition, the epigenetic factors were significantly increased in apoptotic cells following exposure to copper and oxidative stress.

When peat forest fires happen, it leads to burn soil and also humic acids as a dominant organic matter contained in peat soil as well as the forest. The structure and properties of humic acids vary depending on their origin and environment, therefore the transformation of humic acid is also diverse. The impacts of the peat fires on peat soil from Central Kalimantan, Indonesia were investigated through the characterization of humic acids, extracted from soil in burnt and unburnt sites. The characterization of humic acids was performed by elemental composition, functional groups, molecular weight by HPSEC, pyrolysate compounds by pyrolysis-GC/MS, fluorescence spectrum by 3DEEM spectrofluorometer, and thermogravimetry. The elemental composition of each humic substance indicated that the value of H/C and O/C of humic acids from burnt sites were lower than that from unburnt sites. The molecular weight of humic acids from burnt sites was also lower than that from unburnt sites. Pyrolysate compounds of humic acids from unburnt sites differed from those of humic acids from burnt soil. The heating experiment showed that burning process caused the significant change in the properties of humic acids such as increasing the aromaticity and decreasing the molecular weight.

The safety of Tinospora cordifolia and its potential to protect against ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells were investigated. To evaluate the safety of T. cordifolia, cell viability and agarose gel electrophoresis were carried out using PC12 cells treated with 0 to 100 μg mL(-1) of methanol extract of T. cordifolia. T. cordifolia extracts did not show cytotoxicity ranging 0 to 100 μg mL(-1). In addition, T. cordifolia extracts significantly increased cell viability at 1 ng, 10 ng and 1 μg mL(-1) concentrations in serum-deprived medium compared to control. To confirm the protective role against UV-induced damage, PC12 cells alone or in the presence of 10 ng, 100 ng, or 1 μg mL(-1) of T. cordifolia extract were exposed to 250, 270 and 290 nm of UV radiation, which corresponded to doses of 120, 150 and 300 mJ cm(-2), respectively. Treatment with T. cordifolia extracts significantly increased the cell survival rate irradiated at 290 nm. In addition, T. cordifolia extracts significantly reduced cyclobutane pyrimidine dimer formation induced by UV irradiation at all wavelengths. In conclusion, T. cordifolia is not toxic and safe for cells. Our findings can support its application as phototherapy in the medical sector.

Carvacrol is a monoterpenic phenol found in essential oils, is considered a safe food additive, and possesses various therapeutic properties. Numerous studies have also deciphered the protective role of carvacrol on various cytotoxicities. We clarify the effects of carvacrol on cadmium-induced apoptosis in PC12 cells. Carvacrol while co-exposed with cadmium for 48 h raised PC12 cell viability in comparison to only cadmium exposed group. The co-exposure increased the cellular glutathione levels and promoted the expression of glutathione reductase. The magnitude of DNA fragmentation caused by cadmium was also ameliorated by carvacrol. Flow cytometry exhibited the apoptosis rate augmented by cadmium was reduced by carvacrol. Western blotting revealed that cadmium and carvacrol co-exposure alleviated the cadmium-induced down-regulations of mammalian target of rapamycin (mTOR), protein kinase B (Akt), nuclear factor kappa-light-chain-enhancer of activated B cells (NFКB), extracellular signal-regulated kinase-1 (ERK-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) expressions. The co-exposure also reversed action of cadmium by suppressing the cleavage of caspase 3 and reducing the cytosolic levels of cytochrome c and apoptosis inducing factor (AIF). Moreover, carvacrol upon co-exposure significantly increased the intracellular metallothionein content. In conclusion, carvacrol strongly reduced cadmium-triggered oxidative stress and caspase-dependent and caspase-independent apoptosis in PC12 cells.

Humic acid (HA) has been implicated as a contributory factor for blackfoot disease, which is an endemic peripheral vascular disease. We investigated the effect of HA on the regulation of endothelial nitric oxide (NO) synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) to evaluate the involvement of eNOS and related factors in peripheral vascular impairment with HA exposure. Treatment of HUVECs with HA induced upregulation of eNOS. This result coincides with those of previous studies. Furthermore this is the first study to report that HA induces upregulation of heat shock protein (Hsp)90α, Hsp90β, eNOS phosphorylation at Ser1177, and eNOS phosphorylation at Thr495, as compared to that in the control. In contrast, treatment with BAPTA, an intracellular Ca(2+) chelator, inhibited upregulation of these proteins induced by HA. This study demonstrates that HA treatment leads to increases in both Hsp90α and Hsp90β proteins and indicates that Hsp90α leads to eNOS phosphorylation at Ser1177 and that Hsp90β leads to eNOS phosphorylation at Thr495, respectively. Upregulation of eNOS, Hsp90α, and Hsp90β in HUVECs is regulated by intracellular Ca(2+) accumulation induced by HA. These results suggest that upregulation of eNOS phosphorylation at Ser1177 and eNOS phosphorylation at Thr495 produce NO and superoxide anions, respectively, resulting in generation of peroxynitrite, which causes impairment of vascular endothelial cells.

Cadmium (Cd2+) is a well studied inducer of cellular necrosis and apoptosis. Zinc (Zn2+) is known to inhibit apoptosis induced by toxicants including Cd2+ both in vitro and in vivo. The mechanism of Zn2+-mediated protection from Cd2+-induced cytotoxicity is not established. In this study, we aimed to understand the effects of Zn2+ on Cd2+-induced cytotoxicity and apoptosis using PC12 cells. Cell viability and DNA fragmentation assays in PC12 cells exposed to Cd2+ and/or Zn2+ revealed that Cd2+ (5 and 10 μmol/L) alone induced significant cell death, and co-exposure to Zn2+ (5, 10, and 100 μmol/L) for 48 h had a protective effect. Assessment of intracellular free sulfhydryl levels and lactate dehydrogenase activity suggested that Cd2+ (10 μmol/L) induced oxidative stress and disrupted cell membrane integrity. Addition of Zn2+ (10 and 100 μmol/L) reduced Cd2+-mediated cytotoxicity. Changes in expression of the apoptotic factors Bax, Bcl-2, Bcl-x, and cytochrome c were measured via western blot and expression of caspase 9 was detected via reverse transcriptase polymerase chain reaction. Western blots showed that Zn2+ (10 and 100 μmol/L) suppressed Cd2+-induced apoptosis (10 μmol/L) by reducing cytochrome c release into the cytosol, and downregulating the proapoptotic protein, Bax. In addition, expression of caspase 9 was lower in Cd2+ (5 μmol/L)-treated PC12 cells when co-treated with Zn2+ (2 and 5 μmol/L). These findings suggest that the effective inhibition of Cd2+-induced apoptosis in PC12 cells by Zn2+ might be due to suppression of mitochondrial apoptosis pathway and inhibition of Cd2+-induced production of reactive oxygen species.

Humic acid (HA), a group of high-molecular weight organic compounds characterized by an ability to bind heavy metals, is normally found in natural water. Although the impairment of vascular endothelial cells in the presence of humic substances has been reported to be involved in some diseases, the mechanisms responsible for this involvement remain unclear. In this study, we examined the cytotoxicity of HA obtained from peatland in Central Kalimantan, Indonesia, to human vascular endothelial cells, as well as the mechanisms behind these effects. It was found that 50 mg/L HA showed cytotoxicity, which we considered to be mediated by apoptosis through the mitochondrial pathway because of an increase in the expression of caspases 6 and 9 in response to HA administration. In addition, this cytotoxicity was enhanced when cells in this experimental system were exposed to oxidative stress, while it was decreased by the addition of vitamin C. Thus, we conclude that the apoptosis induced by HA depends upon oxidative stress. Furthermore, an iron chelator, DFO, showed a tendency to decrease HA-induced cytotoxicity, suggesting that iron may potentially mediate HA-induced oxidative stress. In conclusion, long-term consumption of HA-rich water obtained from our study area may cause damage to endothelial cells and subsequent chronic health problems.

Alkyl esters of p-hydroxybenzoic acids (parabens), an endocrine disrupter, are used as preservatives in cosmetics and foods. In this study, to understand the relationship between parabens and differentiation in infants, the effects of parabens on apoptosis induced by serum deprivation in PC12 cells were investigated. In addition, apoptosis-related factors were assayed. As results, a tendency toward enhancement of apoptosis was observed in the cells cultured in the serum-free medium with methylparaben, and this tendency was suggested to be related to the contents of BAD, a pro-apoptotic protein. Butylparaben did not show any tendency to enhance apoptosis.

Copper, an essential trace element, induces apoptosis in mammalian cells. However, the precise mechanism of copper-induced apoptosis is still unclear. In this study, to determine the apoptotic pathway initiated by copper treatment, apoptotic factors such as Bax, Bad and Bcl-2, and the caspase family in PC 12 cells treated with copper were measured by Western blot and RT-PCR analyses. The expression of Bax, Bad, cytochrome c and caspases 3 and 9 were increased by copper treatment. From these results, two pathways for copper-induced apoptosis were suggested. At first, an increase of Bax induces the release of cytochrome c from the mitochondria into the cytoplasm owing to binding to apoptotic activating caspase 9 leading to the activation of caspases 3. In the other pathway an increase of Bax and reactive oxygen species activates the release of AIF from the mitochondria. The AIF induces apoptosis via a caspase-independent pathway.

The effects of some methyl-containing compounds added to a choline-deficient diet on the metallothionein mRNA level in the rat liver were studied. The addition of choline or carnitine to the choline-deficient diet did not induce a gain in body weight, while the addition of either betaine or methionine to the choline-deficient diet, or of methionine to the choline-deficient diet with choline significantly increased the body weight. The metallothionein mRNA level in the liver of rats fed on the choline-deficient diet was similar to that of rats fed on the choline-deficient diet with choline, betaine or carnitine. However, the addition of methionine to the choline-deficient diet with or without choline caused a marked suppression in the metallothionein mRNA level in the liver. It is thus surmised that the metallothionein mRNA level in the liver might be regulated by the dietary content of methionine.

2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) is an endocrine disrupter that exerts cytotoxic effects on organisms. In this study, the influence of 2,4,5-T at low concentrations on apoptosis in PC 12 cells was investigated. Although no apoptotic features were observed in PC12 cells treated with 2,4,5-T, it inhibited the DNA fragmentation induced by serum deprivation. In addition, the cell viability of PC12 cells increased after treatment with 2,4,5-T. In conclusion, 2,4,5-T suppressed the apoptosis of the cultured cells. Since apoptosis is a morphological and biochemical description of a physiological mechanism of cell death that is commonly associated with programmed events necessary for development of individuals and organs, the inhibitory effect of 2,4,5-T on apoptosis might cause serious damage to cell homeostasis and differentiation.

Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR. Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity.

The sequence of six amino acid residues -Ser-Cys-Cys-Ser-Cys-Cys- is present in all mammalian metallothionein sequences and has been highly conserved during evolution, although the metallothioneins have divergent primary sequences. To determine whether two serines in the sequence play a crucial role in metal-binding of metallothioneins, a mutant metallothionein with these two serines replaced by leucines was obtained using an Escherichia coli expression system. The expressed protein was analyzed for its chemical and spectroscopic properties. It was confirmed that the mutant metallothionein (MT) bound cadmium through a metal-thiolate complex and that there was no strong difference between the mutant and the wild-type MTs in retaining the metal-binding cluster. However, the metal-binding cluster of the mutant metallothionein was more unstable than that of the wild-type metallothionein. The two conservative serines could play a role in the stability of metal-binding ligands.

The presence of metallothionein (MT) and its mRNA in the ovaries of laying hens is reported. In laying hens injected with cadmium (Cd), Cd accumulated in the follicle walls of the ovaries not in the follicle yolks. In the follicle walls, (Cd, Zn)- MT and its mRNA were found. This indicates that MT bound to Cd is biosynthesized in the follicle walls. We suggest that MT might play a role in sequestering Cd. On the other hand, zinc (Zn) was found in the follicle walls and yolks in the Cd-treated laying hens. The relationship between Zn and (Cd, Zn)-MT is also discussed.

A genetic approach was undertaken to investigate the physiological roles of human metallothionein-2. A constructed expression plasmid, pEXPMTII, in which human metallothionein-IIA cDNA was inserted downstream of a tryptophan-lactose promoter, was used to transform Escherichia coli JM105 strain. Cadmium-binding metallothionein was successfully expressed in E. coli in the medium containing cadmium, while copper and zinc-metallothioneins were scarcely observed in copper- or zinc-containing medium. The amino acid composition and sequence of the biosynthesized cadmium-metallothionein were analyzed. The selectivity of metals bound to metallothionein and the stability of metal-binding forms of metallothionein in E. coli were discussed. In addition, cadmium, zinc, or copper resistance of the cells expressing metallothionein was examined. Cells transformed with the plasmid pEXPMTII and cultured in a medium containing cadmium exhibited tolerance only to cadmium. It was demonstrated that human metallothionein-2 functioned for cadmium detoxification in E. coli.

In this study, to examine the mechanism of diethyl phthalate toxicity to cells, the effects of diethyl phthalate on apoptosis in a PC12 cell system were investigated by assaying apoptotic factors such as caspase-3, Bax, cytochrome c and DNA damage. Diethyl phthalate was shown to enhance the apoptosis induced by serum deprivation according to the results of DNA electrophoresis and TUNEL signal assays, although it could not induce apoptosis itself in the cells. This enhancement was thought to be because of an increase in caspase-3-like activity. In addition, the expression of bax and contents of cytochrome c in the cytosol showed a tendency to increase the cells exposed to diethyl phthalate. These results indicated that diethyl phthalate, a potential endocrine disrupter, affects the apoptotic system in PC12 cells. Diethyl phthalate may enhance oxidative stress such as that induced by reactive oxygen species in PC12 cells.

An endocrine disrupter, bisphenol-A is widely used in the production of plastics and coatings. Recently, it was reported that bisphenol-A affected neurotransmitters in the mammalian brain. On the basis of these reports, it was considered that bisphenol-A affected neuronal differentiation. In this study, the morphological changes in nerve growth factor (NFG)-induced differentiation caused by bisphenol-A were confirmed using a PC12 cell system. When a low concentration of bisphenol-A was added to medium containing NGF, it inhibited neurite extension. In addition, to clarify whether bisphenol-A affects the early and late stages of the NGF-signaling pathway in cell differentiation, changes of phosphorylation of MAP kinases and cAMP-response element binding protein (CREB) in PC12 cells treated with and without BPA in medium containing NGF were investigated using western blot analysis. As results, bisphenol-A significantly inhibited phosphorylation of CREB and ERK1/2 MAPK.

The effects of gamma-hexachlorocyclohexane, one of the endocrine disrupters, widely used for agricultural and medicinal purpose, on apoptosis in PC12 cells were investigated using western blotting analysis and reverse transcriptase-DNA polymerase chain reaction (RT-PCR) method. Apoptosis is a fundamental process necessary for development of individuals and organs. Although gamma -HCH at high concentration did not have an effect on cell viability and apoptosis, DNA fragmentation was slightly enhanced, and apoptotic factors; Bax, Bad, cytochrome c and caspase-3 showed tendency to increase by the addition of a low dose of gamma-HCH to the cell medium. However these changes were not statistically significant. It was concluded that gamma -HCH did not affect on apoptosis in the PC12 cell line system, although gamma -HCH has been reported to induce apoptosis in other cell lines.

In Java and Kalimantan in Indonesia, river water plays important roles in human life; for example, for transportation, and economic activities of the inhabitants. However, industrial, agricultural and domestic water is discarded into rivers directly in many developing countries, including Indonesia, since drainage systems have not been completely constructed. In this study, to evaluate the water quality and to compare those levels of environmental contaminants in developing and developed countries, water quality and contents of endocrine disrupters were measured in a total of 64 water samples (Indonesia; 28 samples and Japan; 36 samples) from 53 sites. The results indicated that, rivers in both capital cities, Jakarta and Tokyo, were contaminated. Water in rivers in Indonesia was not so heavily polluted as in Japan. Pollution in the river water in Indonesia appeared to be caused by the lack of sewerage systems. In addition, the findings on endocrine disrupters indicated that the concentration of alkylphenol in water samples was large enough to affect living organisms.

Lignophenol, prepared using a phase-separation system, is a derivative of lignin, which is one of the components in the plant cell wall, and possesses high phenolic function, high stability and antioxidant properties. However, little is known about the beneficial effect of lignophenol. In this study, we investigated the protective effect of lignophenol from the beech tree (Fagus crenata Blume) on copper- and zinc-mediated apoptosis in PC12 cells by using DNA fragmentation and TUNEL assays. In DNA fragmentation assays, the DNA ladder patterns in the PC12 cells treated with 200 microM Cu and 200 microM Zn were enhanced, whereas the DNA ladder pattern was hardly observed in these cells treated with 20 mM lignophenol. In the TUNEL assay, TUNEL signals increased significantly in the untreated PC12 cells exposed to 200 microM Cu compared with the control. In contrast, the degree of apoptosis in the 20 mM lignophenol-treated cells was significantly lower than in the untreated cells, indicating that lignophenol inhibited Cu-induced apoptotic cell death in PC 12 cells. In the 200 microM Zn-exposed group, the degree of apoptosis in the 20 mM lignophenol-treated cells was also low compared with the untreated cells. In conclusion, these results suggest that lignophenol plays a role in protecting against Cu- and Zn-mediated PC12 apoptotic cell death.

Metallothionein-3/growth inhibitory factor is a brain-specific member of the metallothionein gene family, and impairs the survival and neurite formation of cultured neurons. Metallothionein-3 can bind heavy metals such as Zn, Cu, or Cd almost in the same way as other metallothionein family. However, its biological function as growth inhibitory factor apparently distinguishes metallothionein-3 from other metallothioneins. To better understanding of the relationship between the growth inhibitory activity of metallothionein-3 and metals bound to metallothionein-3, the metal-binding ability of metallothionein-3 was analyzed in comparison with those of metallothionein-1 and -2. The metal-binding ability of metallothionein-3 was evaluated by pH titration and 5-5' dithiobis (2-nitrobenzoic) acid (DTNB) analysis as compared with those of the other metallothioneins. The affinity of metal ions for metallothionein-3 was indicated as follows, Cu>Cd>Zn, same as metallothionein-1 and -2. However, the affinity of metallothionein-3 to Cu was much higher than that of metallothionein-1 and -2. The strong affinity to Cu of metallothionein-3 might be related to its growth inhibitory activity.

Although nonylphenol is well known as an endocrine disrupting chemical, there is little information concerning biological effect of nonylphenol. In this study, we investigated effect of nonylphenol on apoptosis induced by serum deprivation in PC12 cells using TUNEL and DNA fragmentation assays. In addition, changes in contents of proapoptotic factors, Bad and Bax, and antiapoptotic factor, Bcl-2, and enzyme activity of caspase-3 were studied. Below 100 ng/ml of nonylphenol increased TUNEL signals, DNA fragmentation and content of proapoptotic factor, Bad as compared to those by serum deprivation without nonylphenol. Furthermore, addition of nonylphenol enhanced caspase-3 activity and Z-VAD, caspase-3 inhibitor, diminished such effect. These results indicated that below 100 ng/ml of nonylphenol enhanced apoptosis induced by serum deprivation via caspase-3 activation in PC12 cell.

The present study was carried out to investigate the effect of gold (Au) injection on copper (Cu) and two types of ceruloplasmin (Cp), total Cp (ID1) and active Cp (ID2), metallothionein (MT) in the serum, kidney and liver, and 8-hydroxydeoxyguanosine (8-OHdG) in the rat kidney. The Cu contents in sera and kidneys of Au-injected rats were 1.7 and 5.5 times higher than those in sera and kidneys of control rats, respectively. The most of Cu in the sera of the control rats or Au-injected rats were observed in the Cp fractions from a Sephacryl S-200 column. The Cu concentration in the Cp fractions was increased by Au injection. Significant increases of ID1 and ID2 were found in the sera of the control rats and Au-injected rats, while there was no significant difference in those concentrations of livers or kidneys between the control rats and Au-injected rats. Our results indicated that the most of Cp existed as active ID1. The immunoreactivity of 8-OHdG was located in the cortex of the Au-injected rat. These results indicated that the oxidative DNA damage occurred in the renal cortex of the Au-injected rat and the localization of DNA damage did not coincide with that of Cu-MT. These findings suggest that the oxidative DNA damage in the kidneys of rats injected with Au is associated with Cu except Cu-MT.

The effect of trichloroethylene (TCE) on long-term potentiation (LTP) was studied using both electrical and optical recording. The hippocampi from mice injected with 300 mg/kg or 1000 mg/kg TCE were sliced 24 h after administration. The field potential from the CAI was recorded. After the application of tetanus, population spikes (PS) were potentiated in all groups, but the post-per-pre ratio of PS was smaller in TCE groups than in the control. Optical recording was also carried out in 1000 mg/kg TCE-injected mice and a new analytical method using a high speed camera was employed. After the induction of tetanus, the optical signal was potentiated in both TCE and control groups. However, the post-per-pre ratio of the optical signals and response area were smaller in the TCE groups than in the control. It was suggested that the impairment of LTP is one of the mechanisms of the impairment of immediate memory after acute exposure to TCE in humans.

This study was undertaken to investigate the effect of taurine on the infiltration of macrophages and the progression of renal interstitial fibrosis in the kidneys of rats treated with cisplatin (CDDP). Male rats in different groups were treated as follows: (1) saline as control, (2) CDDP and (3) CDDP plus taurine in drinking water. At weeks 2, 4 and 6 after the fifth CDDP injection, we examined platinum content, the kinetics of macrophages and the areas of fibrosis. In the histologic analyses, fibrotic areas were found to have developed around dilated or atrophic tubules in the corticomedullary junction in the kidneys of CDDP-treated rats, while CDDP-plus-taurine-treated rats showed a reduction of the extent and magnitude of damage. These findings reflect the fact that the percentage of fibrotic areas in the kidney of CDDP-plus-taurine-treated rats was significantly lower than in that of CDDP-injected rats throughout the recovery period (P

Increased use of motor vehicles has produced various risks to human health due to air pollution by noxious gases, heavy metals and roadside dust. Since the late 1970s, the wide spread use of studded tires for cars has caused pavement wear, resulting in not only economic losses, but also roadside air pollution in cold and snowy regions in Japan. The most serious environmental problem in Sapporo, a city with heavy snowfall, in the 1980s, was roadside dust derived from studded tires. The inhabitants suffered from this dust in the early winter and in the early spring when the streets were not covered with snow. To investigate the influence of such roadside dust upon human health, particle numbers in classified sizes of roadside dust were counted after the roadside dust in the air was collected with a device we constructed at 30, 60, 90, 120, 150, and 180 cm above the pavement surface. The results indicated that the concentration of roadside dust in the air did not greatly vary according to the height from the pavement surface. The results also suggested that xenogranuloma, reported in lungs of stray dogs, under roadside dust-pollution conditions such as those examined here, may occur in humans in the future.

1. Activities, contents and true specific activities of superoxide dismutase (SOD) were determined in human erythrocytes of 105 normal healthy subjects. 2. The mean SOD activity assayed by the inhibition of xanthine autoxidation was 11·0 × 103 units/g of haemoglobin (Hb). 3. The mean SOD content assayed by an immunochemical method was 456 μg/g of Hb. 4. Both activity and content of SOD showed normal distributions, and no significant variations with regard to sex and age were detected. 5. A highly positive correlation between activities and contents of SOD was observed in normal healthy subjects (r = 0·77, P < 0·001). 6. True specific activity was calculated from levels of activity and content of SOD. The mean true specific activity of SOD in human erythrocytes was 23·7 units/μg of SOD. 7. There was no significant difference in true specific activity between young and old subjects.

Levels of human erythrocyte catalase activity were determined in 38 patients with thyroidal dysfunction. In patients with hyperthyroidism, erythrocyte catalase activities were found to be higher than the levels of normal subjects (P less than 0.001). In hypothyroidism, erythrocyte catalase activities were of the same order as those of normal subjects. Significantly high positive correlation was found between erythrocytes catalase activity and the levels of thyroxine (r = 0.5794, n = 36, P less than 0.001), and slight positive correlation was detected between catalase activity and the levels of triiodothyronine (r = 0.3978, n = 33, P less than 0.05). A decreased erythrocyte catalase activity was observed when erythrocytes lysate was incubated with thyroid hormones. It was suggested that erythrocyte catalase activity had close relationship with thyroid state, however, direct effect of thyroid hormones were not observed on erythrocyte catalase assay system in vitro.