4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induced lung tumors in rodents and is likely involved in human lung cancer. 4-(Hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (HO-methyl NNK) glucuronide, a glucuronide of the reactive intermediate of NNK, has been identified in rats. The aim of this study is to estimate the role of HO-methyl NNK glucuronide in the tumorigenic effects of NNK. We investigated the urinary excretion and tissue distribution of HO-methyl NNK glucuronide in A/J mice, which are susceptible to NNK carcinogenesis, and C57BL/6J mice, which are resistant to NNK carcinogenesis. The cumulative urinary excretion of the HO-methyl NNK glucuronide in the C57BL/6J mice was more than 20 times higher than in the A/J mouse urine. Tissue concentrations of HO-methyl NNK glucuronide were also higher in the C57BL/6J mice than in the A/J mice. Assessment of the stability of HO-methyl NNK glucuronide in liver homogenates at physiological pH conditions showed that more than 60% of the glucuronide remained until 2 hr of incubation. These results suggested that HO-methyl NNK glucuronide is likely to be a detoxified metabolite and could be one reason for differences in the susceptibility to NNK tumorigenesis between the two strains. Once HO-methyl NNK is formed in tissues, C57BL/6J mice have a high ability to form HO-methyl NNK glucuronide so that HO-methyl NNK, the reactive intermediate formed from NNK, is readily excreted in urine as a stable form.
New five P-III snake venom metalloproteinases (SVMPs): EpyB2 (62 kDa), EpyB3 (62+23 kDa), EpyB4 (60 kDa), EpyB5 (67 kDa) and EpyB6 (66 kDa) of the most dangerous viper, Echis pyramidum pyramidum (Epy), were purified and characterized in a set of biochemical assays. The SVMPs were purified by applying a protocol of two successive chromatographic steps. Three purified SVMPs “EpyB2, EpyB4, and EpyB5” have hemorrhagic activity with MHDs, 7 μg, 7.6 μg and 15 μg, respectively; furthermore, they have high preference towards fibronectin, collagen, gelatin, fibrin and hemoglobin substrates compared with non-hemorrhagic SVMPs (EpyB3 and EpyB6). All the purified SVMPs showed remarkable thermal and pH stability, inhibited by metalloproteinase inhibitors and Zn2+, Mn2+, Ni2+, Co2+, Cu2+, and Hg2+. The purified SVMPs act as α-fibrinogenases, prothrombin activators and procoagulants. In conclusion, Epy venom has multiple SVMPs that are responsible for hemorrhagic events and thus represent a significant health hazard for victims of envenomation, however, they may be useful for treating diseases involving abnormal blood clot formation.
Generally, reactive metabolites are detoxified by conjugation with glutathione (GSH). A GSH-depleted model was prepared by administering L-buthionine-(S,R)-sulfoximine (BSO), which can be used to detect hepatic damage by reactive metabolites. However, BSO may cause adverse effects on other organs, such as renal damage by reactive metabolites because it depletes GSH in the whole body. The present study was designed to examine whether it was possible to specifically detect hepatic damage by reactive metabolites without reducing renal GSH levels by administering BSO in a time course when hepatic GSH levels are naturally reduced. Male BALB/c mice were administered reverse osmosis (RO) water or 20 mmol/l BSO in drinking water for 4 days. Subsequently, animals in the RO water group were orally administered 500 mg/kg acetaminophen (APAP) at 9:00 or 15:00 and in the BSO group at 9:00 for 4 days. As a result, severe hepatic damage and necrosis of the renal proximal tubules were observed in the BSO/APAP administration at 9:00 group, and all animals died on 1 or 2 days after APAP administration. Hepatic damage was clearly increased in the RO water/APAP administration at 15:00 group compared with the RO water/APAP administration at 9:00 group. However, renal damage and deaths were not observed. This BSO administration model may detect renal damage induced by reactive metabolites. Using an administration time course, whereby hepatic GSH levels were naturally reduced, hepatic damage by reactive metabolites can be detected without secondary renal effects.
2-Cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid methyl ester (CDDO-Me; bardoxolone methyl) is one of the synthetic oleanane triterpenoids (SOs). It is known that it is the strongest Nrf2/ARE signaling inducer of SOs and slightly inhibits immune response. Little was known about the immunomodulatory action of CDDO-Me in vivo. We assessed its immunosuppressive potential by using the modified mouse lymph node assay (LLNA) including immunosuppression-related gene expression analysis. In the modified LLNA, CDDO-Me showed a significant decrease in lymph node weight and changes in expressions of the immunosuppression-related genes, Zfp459 and Fmo2. It has been already reported that a decrease in lymph node weight was induced by several types of immunosuppressive chemicals such as calcineurin inhibitors, antimetabolites, steroids, and alkylators. In addition, changes in Zfp459 and Fmo2 expression was reported in response after only treatment of antimetabolites. From these results, CDDO-Me is considered to have an immunosuppressive action and similar mechanism to antimetabolites.
Particulate air pollutants and mineral fibers activate inflammatory cells to release oxidants, which contribute to inflammation and injury in the lower respiratory tract. Our aim was to compare the role of silica particle size with mineral fiber length and width in the ability to induce superoxide release from rat alveolar macrophages. We estimated the ability of four types of silica particle samples, with different mode diameter, and three types of mineral fiber samples, with different geometric mean lengths and widths, to induce lucigenin-dependent chemiluminescence (CL) from the cells per number of dust particles (i.e., silica particles and mineral fibers). A close positive correlation was observed between dust size and the ability to induce CL in silica as well as mineral fiber samples. Moreover, the ability of silica samples to induce CL was weaker than that of long mineral fiber sample. This ability increased at a larger rate in small silica particle and thin mineral fiber samples than in large silica particle and thick mineral fiber samples at the initial stage of administration. These results suggest that the kinetics of the induction superoxide release from macrophages is similar between silica particles and mineral fibers; moreover, this depends on silica particle size and mineral fiber geometry. Finally, large silica particles were more active than small ones.
This study examined the relationship between neurobehavioral changes and alterations in gene expression profiles in the brains of mice exposed to different levels of Hg0 during postnatal development. Neonatal mice were repeatedly exposed to mercury vapor (Hg0) at a concentration of 0.057 mg/m3 (low level), which was close to the current threshold value (TLV), and 0.197 mg/m3 (high level) for 24 hr until the 20th day postpartum. Behavioral responses were evaluated based on changes in locomotor activity in the open field test (OPF), learning ability in the passive avoidance response test (PA), and spatial learning ability in the Morris water maze (MM) at 12 weeks of age. No significant differences were observed in the three behavioral measurements between mice exposed to the low level of Hg0 and control mice. On the other hand, total locomotive activity in mice exposed to the high level of Hg0 was significantly decreased and central locomotion was reduced in the OPF task. Mercury concentrations were approximately 0.4 μg/g and 1.9 μg/g in the brains of mice exposed to the low and high levels of Hg0, respectively. Genomic analysis revealed that the expression of 2 genes was up-regulated and 18 genes was down-regulated in the low-level exposure group, while the expression of 3 genes was up-regulated and 70 genes was down-regulated in the high-level exposure group. Similar alterations in the expression of seven genes, six down-regulated genes and one up-regulated gene, were observed in both groups. The results indicate that an increase in the number of altered genes in the brain may be involved in the emergence of neurobehavioral effects, which may be associated with the concentration of mercury in the brain. Moreover, some of the commonly altered genes following exposure to both concentrations of Hg0 with and without neurobehavioral effects may be candidates as sensitive biomarker genes for assessing behavioral effects in the early stages of development.
Maternal exposure to environmental factors is implicated as a major factor in the development of the immune system in newborns. Newborns are more susceptible to microbial infection because their immune system is immature. Development of lymphocytes reflects an innate program of lymphocyte proliferation. The aim of this study was to investigate the effects of maternal exposure to carbon black nanoparticle (CB-NP) during early gestation on the development of lymphoid tissues in infantile mice. Pregnant ICR mice were treated with a suspension of CB-NP (95 μg kg-1 time-1) by intranasal instillation on gestational day 5 and 9. Spleen tissues were collected from offspring mice at 1, 3, 5, and 14 days postpartum. Splenocyte phenotypes were examined by investigating the pattern of surface molecules using flow cytometry. Gene expression in the spleen was examined by quantitative RT-PCR. CD3+ (T), CD4+ and CD8+ cells were decreased in the spleen of 1-5-day-old offspring in the treated group. Expression level of Il15 was significantly increased in the spleen of newborn male offspring, and Ccr7 and Ccl19 were increased in the spleen of female offspring in the CB-NP group. Splenic mRNA change profiles by CBNP were similar between male and female offspring. This article concluded that exposure of pregnant mothers to CB-NP partially suppressed the development of the immune system of offspring mice. The decrease in splenic T cells in the treated group recovered at 14 days after birth. This is the first report of developmental effect of nanoparticle on the lymphatic phenotype.
As drug-induced seizures have severe impact on drug development, evaluating seizure induction potential of candidate drugs at the early stages of drug discovery is important. A novel assay system using zebrafish has attracted interest as a high throughput toxicological in vivo assay system, and we tried to establish an experimental method for drug-induced seizure liability on the basis of locomotor activity in zebrafish. We monitored locomotor activity at high-speed movement (> 20 mm/sec) for 60 min immediately after exposure, and assessed seizure liability potential in some drugs using locomotor activity. However this experimental procedure was not sufficient for predicting seizures because the potential of several drugs with demonstrated seizure potential in mammals was not detected. We, therefore, added other parameters for locomotor activity such as extending exposure time or conducting flashlight stimulation (10 Hz) which is a known seizure induction stimulus, and these additional parameters improved seizure potential detection in some drugs. The validation study using the improved methodology was used to assess 52 commercially available drugs, and the prediction rate was approximately 70%. The experimental protocol established in this present study is considered useful for seizure potential screening during early stages of drug discovery.
Dihydropyrazines (DHPs) are glycation products that are nonenzymatically generated in vivo and in food. In this study, we compared the effects of 2,3-dihydro-5,6-dimethylpyrazine (DHP-1), a low toxicity DHP, and 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), a high toxicity DHP on the redox indices in HepG2 cells. An apparent increase in intracellular hydrogen peroxide concentration was observed at 24 hr after 1 mM DHP-3 treatment. In addition, DHP-3 exposure significantly increased the mRNA levels of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC), which are stress-responsive genes, at 6 hr (HO-1 and GCLC), 12 hr (HO-1 and GCLC) and 24 hr (GCLC) after exposure. These indices, with the exception of the increase in GCLC mRNA after a 6 hr exposure, were not affected by treatment with 1 mM DHP-1. HO-1, GCLC, and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels also increased at 6 hr (Nrf2), 12 hr (Nrf2, HO-1 and GCLC) and 24 hr (GCLC) after DHP-3 treatment. The increase in HO-1 and Nrf2 protein levels were observed with lower concentration (0.5 mM) of DHP-3, and in agreement with this, antioxidant responsive element-luciferase reporter activity was significantly increased with exposure to at least 0.5 mM DHP-3. These results support our previous report establishing that oxidative stress is in part involved in the effects of DHP on mammalian cells. Additionally, our results suggest that the cell response to DHP-3 exposure was exerted via the activation of the Nrf2-ARE signal pathway.
Betel-quid use is associated with the risk of liver cirrhosis and hepatocellular carcinoma. The aim of the present work was to evaluate the impact of arecoline on human hepatic cytochrome P450 (CYP) enzymes in vitro and rat hepatic CYP enzymes, as well as the hepatic oxidative stress and liver injury of rats in vivo. The in vitro results indicated that arecoline hydrobromide (AH) has no significant effect on the activities of CYP2B, 2C9, 3A4, 1A2, 2E1 and 2D6 in human liver microsome (HLM). However, oral administration of AH at 4 and 20 mg/kg/d for seven consecutive days significantly increased the activities of rat hepatic CYP2B, 2E1, 2D, 3A, 2C and 1A2. In addition, AH at 100 mg/kg/d significantly increased the levels of ALT, AST and MDA, decreased the levels of SOD, CAT, GSH-Px and GSH, in rat liver. The in vivo induction of AH on rat hepatic CYP isoforms suggested that the high risk of metabolic interaction should be existed when the substrate drugs of the six kinds of CYP isoforms was administered in betel-quid use human. Furthermore, the in vivo results also suggested that AH-induced hepatoxicity should be associated with the induction of AH on rat hepatic CYP2E1 and 2B.
Chemically induced depigmentation of the skin, which occurs following exposure (application or inhalation) to a depigmenting agent, is a disease with clinical findings similar to vitiligo. Recently, skin depigmentation possibly resulting from exposure to 4-(4-hydroxyphenyl)-2-butanol (HPB) was reported in humans. However, the role of HPB as the causative material of this skin depigmentation was not clear. To evaluate whether HPB has the potential for skin depigmentation, we characterized its effects on the skin of pigmented guinea pigs. Following exposure to 30% HPB 3 times/day for about 20 days, we found that obvious skin depigmentation was induced in brown and black guinea pigs. In the depigmented skin, there was a marked reduction in melanin pigment, and decreased numbers of DOPA and S-100 positive epidermal melanocytes were observed histologically. In addition, the depigmentation gradually recovered spontaneously and the number of melanocytes in the skin also increased after terminating the application of HPB. Complete re-pigmentation needed 31 to 70 days to return to the original baseline level. These data indicate that skin depigmentation is induced by the toxicity of HPB to epidermal melanocytes, and that the induced skin depigmentation can recover by terminating the application of HPB.
According to a recent study, mercury (Hg) exposure contributes to Alzheimer’s disease (AD). However, the underlying mechanisms are not understood. This study investigated the effect of methylmercury (MeHg) treatment on the generation, degradation, and transport of amyloid β-protein (Aβ) in the brain. Wistar rats were administered MeHg by gavage (0, 20, 200, and 2,000 μg Hg/kg/day) for 4 weeks. The total Hg in the blood and brain regions was measured, and the levels of Aβ42 in plasma, cerebrospinal fluid (CSF), and brain regions were estimated. The expression of amyloid precursor protein (APP), beta-site APP-cleaving enzyme 1 (BACE1), and neprilysin (NEP) in the brain regions was determined, in addition to the expression of low-density lipoprotein receptor-related protein 1 (LRP1) and the receptor for advanced glycation end products (RAGE) in the brain capillary endothelium (BCE). Finally, the amount of soluble low-density lipoprotein receptor-related protein (sLRP) in the plasma was determined. Aβ42 levels were decreased in the CSF of the 2,000 μg Hg/kg/day group compared with controls, and Aβ42 levels increased in the hippocampus (HC) in a dose-dependent manner. MeHg decreased LRP1 expression but increased RAGE levels in BCE. sLRP levels were decreased in the plasma of the MeHgtreated rats. They were positively correlated with CSF Aβ42 and negatively correlated with Aβ42 and Hg levels in HC. These results imply that MeHg reduces the transportation of Aβ, thereby resulting in the accumulation of the protein in the HC. Plasma sLRP levels may be an early biomarker of Hg-induced Aβaccumulation in the brain.
Background: Indoxyl sulfate is considered to play a pathological role in the progression of chronic kidney disease. The aim of this study was to investigate the deleterious effects of indoxyl sulfate on kidney mesangial cells. Materials and Methods: Rat renal mesangial cells were exposed to indoxyl sulfate at a serial concentrations. Cytotoxicity of indoxyl sulfate on renal mesangial cells was determined using MTT assay. Protein levels of cleaved caspase-3, angiotensin, angiotensin converting enzyme (ACE) and renin were detected by immunoblotting. Reverse transcriptional PCR was performed to determine the mRNA expression. Results: Level of cleaved caspase-3 was augmented while the cell viability was inhibited by indoxyl sulfate in a dose-dependent manner. The mRNA expressions of pro-renin and ACE were upregulated in mesangial cells exposed to indoxyl sulfate. Level of renin and ACE was increased in response to indoxyl sulfate exposure in time-dependent fashion. Conclusion: Indoxyl sulfate increased viability and induced cell death of renal mesangial cells, which is time-dependent. The loss of cell viability is attributed to caspase-3 activity through apoptosis pathway. RAS in renal mesangial cells is activated in response to indoxyl sulfate treatment.
Aflatoxin B1 (AFB1) airway inhalation represents an additional route of exposure to this toxin. However, the association between AFB1 inhalation and serum AFB1 albumin adducts remains unclear. The aim of this study was to explore the association between airway exposure to AFB1 and serum AFB1 albumin adduct concentrations via an epidemiological study, as well as in an AFB1 airway exposure animal model. Our epidemiological study was conducted in a sugar factory in the Guangxi Autonomous Region of China. In order to examine fungal contamination, air samples were obtained in the workshop and areas outside the workshop, such as the office and nearby store. Dust samples were also collected from the bagasse warehouse and presser workshop, and were analyzed using an indirect competitive enzyme-linked immunosorbent assay (ELISA). Additionally, blood samples were collected from a total of 121 workshop workers, and a control group (n = 80) was comprised of workers who undertook administrative tasks or other work outside the workshop. The animal experiment was conducted in the laboratory animal center of Guangxi Medical University, where a total of 60 adult male rabbits were involved in this study. By intubation, AFB1 was administered in three groups of rabbits daily, at dose rates of 0.075, 0.05 and 0.025 mg/kg/day for a period of 7 days. Blood samples were collected on day 1, day 3, day 7 and day 21, and the measurements of the AFB1 albumin adducts in the serum were performed by a double antibody sandwich ELISA. The epidemiological study showed that serum albumin adducts were detected in 67 workshop workers (55.37%), and the values ranged 6.4 pg/mg albumin to 212 pg/mg albumin (mean value: 51 ± 4.62 pg/mg albumin). In contrast, serum albumin adducts were detected in only 7 control group participants, with the values ranging from 9 pg AFB1/mg albumin to 59 pg/mg albumin (mean value: 20 ± 13.72 pg/mg albumin). The animal experiment revealed that the rabbits had detectable levels of AFB1 in their serum with a minimum effective dose of 0.05 mg/kg/day; while 11 of 17 (64.71%) rabbits had detectable levels of AFB1 albumin adducts in the high exposure group (0.075 mg/kg/day), and only 5 rabbits (26.32%) had detectable levels of AFB1 albumin adducts in the moderate exposure group (0.05 mg/kg/day). No rabbits had detectable levels of AFB1 albumin adducts in the low exposure group (0.025 mg/kg/day). Our results demonstrated that only exposure to a certain level of AFB1 would result in detectable levels of serum AFB1 albumin adducts. Interventional programs aimed at reducing exposure to AFB1 by inhalation are urgently needed in high-risk populations. Additional large-sample, well-designed randomized controlled trials are needed to further confirm our results.
One of the mechanisms of phototoxicity is photo-reaction, such as reactive oxygen species (ROS) generation following photo-absorption. We focused on ROS generation and photo-absorption as key-steps, because these key-steps are able to be described by photochemical properties, and these properties are dependent on chemical structure. Photo-reactivity of a compound is described by HOMOLUMO Gap (HLG), generally. Herein, we showed that HLG can be used as a descriptor of the generation of reactive oxygen species. Moreover, the maximum-conjugated π electron number (PENMC), which we found as a descriptor of photo-absorption, could also predict in vitro phototoxicity. Each descriptor could predict in vitro phototoxicity with 70.0% concordance, but there was un-predicted area found (gray zone). Interestingly, some compounds in each gray zone were not common, indicating that the combination of two descriptors could improve prediction potential. We reset the cut-off lines to define positive zone, negative zone and gray zone for each descriptor. Thereby we overlapped HLG and PENMC in a graph, and divided the total area to nine zones with cut-off lines of each descriptor. The rules to prediction were decided to achieve the best concordance, and the concordances were improved up to 82.8% for selfvalidation, 81.6% for cross-validation. We found common properties among false positive or negative compounds, photo-reactive structure and photo-allergenic, respectively. In addition, our method could be adapted to compounds rich in structural diversity using only chemical structure without any statistical analysis and complicated calculation.
Emphysema can be induced in animals by postnatal treatment with dexamethasone (Dex) and such models have been widely used for various research. However, it is not clear what are the effects of Dex on assembly of alveolar elastic fibers in the emphysema model in mice. This study compared the expression profile of genes related to alveolar development between Dex treated and control mice during the treatment from postnatal day 3 (P3) to P14 with a 2-day break. From morphological observation of lung sections on P42, we confirmed the induction of emphysema in the treated mice. The mRNA expression level of fibrillin-1, which consists of microfibrils as a scaffold to form elastic fibers, and fibulin-5, which is a key protein reinforcing the fibers, reached maximum on P7 in control mice. However, in the Dex group, expression levels both types of mRNA were much lower with no clear expression peak. On the other hand, mRNA expression of tropoelastin, the main component in elastic fibers, reached maximum on P5 in the Dex group, which was 9 days earlier than in the control group. At this time, the amount of microfibrils might not be enough for tropoelastin to be deposited completely in Dex treated mice. This imbalance in the expression of tropoelastin and microfibril might interfere with the efficient formation of elastic fibers.
Environmental levels of bisphenol A (BPA) are a global concern because the compound can cause damage to reproductive organs, the thyroid gland, and brain tissues at developmental stages. Plants are important in removing BPA from the atmosphere, soil, and water. However, knowledge on the mechanism by which plants respond to this compound is limited. To determine the response mechanism of plants to BPA, we used a microarray system to analyze the gene expression patterns of Arabidopsis thaliana after irrigation with 3.0 mM BPA. We identified 651 genes that were differentially expressed upregulated and 470 genes that were downregulated by BPA. These genes may specifically contribute to BPA uptake, transformation, conjugation, and compartmentation in plants. The potential function of upregulated genes in plant defense against BPA was also determined.
J. Toxicol. Sci., 37, 711-721 (2012).Error : Data for gpt and Spi- MFs in the lungs of male and female gpt delta rats treated with safrole for 13 weeks are summarized in Tables 7 and 8, respectively.-> Correction : Data for gpt and Spi- MFs in the lungs of male and female gpt delta rats treated with 1-MN for 13 weeks are summarized in Tables 7 and 8, respectively.