Malondialdehyde-modified low-density lipoprotein (MDA-LDL) and oxidized LDL (Ox-LDL), which accelerate the pathogenesis of arteriosclerosis, are thought to be involved in parthenogenesis caused by smooth muscle cell proliferation. In this study, we investigated the suppression mechanism of polycyclic aromatic hydrocarbons (PAHs) on the growth of an MDA-LDL-induced human acute monocyte leukemia suspension cell line (THP-1 cells). We found that PAHs suppressed MDA-LDL-induced THP-1 cell growth. Cotreatment with benzo[a]pyrene (BaP) or 3-methylchoranthrene (3-MC) decreased MDA-LDL-induced THP-1 cell growth, whereas treatment with benzo[e]pyrene (BeP) or pyrene, which is not a ligand for the arylhydrocarbon receptor (AhR), did not decrease THP-1 cell growth. Our findings clearly demonstrated that THP-1 cell growth, which was suppressed by PAHs, was restored by the addition of α-naphtoflavone, which is a partial antagonist to AhR. Moreover, it was shown that cotreatment with MDA-LDL and BaP markedly induced the expression of human cytochrome P4501A1 (hCYP1A1) messenger ribonucleic acid (mRNA) and significantly induced the expressions of p53 and p21 mRNAs. In support of these findings, AhR small interfering RNA suppressed the induced level of p21 mRNA and by BaP and the overexpression of hCYP1A1 significantly induced levels of p21 mRNA. On the other hand, the uptake rate of [14C]BaP into cells was increased more significantly by cotreatment with MDA-LDL than by treatment with [14C]BaP alone. These results strongly suggest that the suppression of MDA-LDL-induced THP-1 cell growth is caused by the increased uptake of PAHs, which strongly activate the AhR signal pathway accompanying DNA damage.
The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.
Drug induced liver injury (DILI) accounts for more than 50% of the cases of acute liver failure in this country, and is the major cause of drug withdrawal from the market. DILI has been associated with the induction of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α). Pro-inflammatory cytokines activate the mitogen activated protein kinase, c-Jun-N-terminal kinase (JNK) in the liver. Recent studies have shown that JNK can regulate the hepatotoxicity of the analgesic, acetaminophen (APAP). Several reports have shown that inflammation induced by the endotoxin, lipopolysaccharide (LPS) augments the toxic response to hepatotoxicants in vivo. However, the mechanism by which inflammation alters drug-induced hepatotoxicity is not known. This study investigated the role of inflammatory mediators in regulating the toxicity of the hepatotoxic drugs, APAP or chlorpromazine (CPZ) in primary mouse hepatocytes. We found that, pre-treatment with TNF-α resulted in ~50 to 60% increase in alanine aminotransferase (ALT) levels by APAP or CPZ, while interleukin-1β (IL-1β) or IL6 treatments showed only 15-20% increase in ALT release. The bacterial components, LPS or lipoteichoic acid (LTA) increased ALT release by ~35 to 38% upon drug treatment of the hepatocytes. The JNK inhibitor, SP600125 significantly diminished APAP and CPZ toxicity with or without TNF-α. Pre-treatment with TNF-α resulted in prolonged activation of JNK (upto 2 hr) in the presence of APAP or CPZ. These results show that TNF-α is the major cytokine involved in sensitizing hepatocytes to APAP- or CPZ-induced hepatotoxicity, likely by a mechanism involving sustained activation of JNK.
This work has been carried out to investigate the conditions which lead to removal of the biogenic amines through the model system. Also, the main goal of this research work is trying to remove biogenic amines; histamine and tyramine, from some Egyptian foods such as tomato, strawberry, banana and mango to prevent their allergy effect. Histamine and tyramine have been affected by pyrogallol, catechol, starch, ascorbic and chlorogenic acids at different levels with different conditions. Some natural additives like glucose, spices, milk, vanillin, starch, orange juice, ascorbic and citric acids, showed an effective effect on disappearance of histamine and tyramine. By studying the effect of some additives on biogenic amines, it was found that tomato showed a decrease in histamine and tyramine concentrations by adding spices. Strawberry and banana showed a clear decrease in histamine and tyramine concentrations by treating them with ascorbic acid. Treating mango by milk led to increase of histamine level while milk with chocolate increases both histamine and tyramine concentrations.
Effects of pregnancy and lactation on warfarin-induced changes in blood coagulation-related parameters were examined in rats. Warfarin (0.5 mg/kg/day) was given orally to pregnant and non-pregnant rats for 3 days from gestation day (GD) 17 to 19 or to lactating and non-pregnant rats for 3 days from post partum day (PPD) 10 to 12. Blood samples were collected from the rats on the day following the last administration (GD 20 or PPD 13) to measure prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombotest (TBT), factor VII and X activities and anti-thrombin III concentration (ATIII). Administration of warfarin to non-pregnant rats resulted in significant prolongation of APTT and TBT and significant decreases in factor VII and X activities. On the other hand, similar but not significant changes were observed in pregnant rats and similar significant but less prominent changes were observed in lactating rats. The reduction of the anticoagulant effects of warfarin may partially be related to high plasma 17β-estradiol concentration in pregnant rats and to high plasma prolactin concentration in lactating rats, respectively.
Occupational exposure to toluene diisocyanats (TDI) may cause asthma. In asthma patients, the allergic syndromes correlate cytokine production with the elevation in cytosolic calcium concentration [Ca2+]c of lymphocytes in airway. We previously found TDI induces calcium signaling in neuronal cells. TDI mainly gets into human body via inhalation; therefore this study investigated the possibility of TDI inducing the changes in [Ca2+]c in airway. We used human lung epithelial cell line H1355, human T-cell line Jurkat, and human neuroblastoma SH-SY5Y cells to present the kinds of cells existing in airway. The changes of [Ca2+]c were measured by Fura-2 fluorescent dye. Results show that TDI induced an elevation in [Ca2+]c in those cell lines and two primary isolated cells, bovine adrenal chromaffin cells and human white blood cells. Cytokine release and their gene expressions of Jurkat cells and human white blood cells were measured by ELISA and reverse transcription polymerase chain reaction. TDI acutely promoted the interleukine-4 (IL-4) release significantly in both Jurkat cells and human white blood cells. TDI-induced IL-4 release was suppressed in the presence of 1,2-bis- (O-aminophenoxy)ethane-N,N,N’,N’- tetraacetic acid (BAPTA), an intracellular Ca2+ chelator, in Jurkat cells. In the hand of gene expression, TDI induced an increase in the mRNA level of TNF-α and IL-4 in Jurkat cells. We conclude that the release of IL-4 were coupled with the elevation in [Ca2+]c induced by TDI. Further studies are required to clarify the roles of TDI-induced IL-4 secretion in acute inflammation.
We examined the sensitivity of metallothionein (MT)-III null mice to cadmium (Cd)-induced acute hepatotoxicity. MT-I/II null mice were also used to compare Cd toxicities between MT-III null mice and MT-I/II null mice. Male MT-I/II null mice, MT-III null mice and wild-type mice were given s.c. injection of Cd (5-20 µmol/kg) and then the blood and liver were collected from each mouse under ether anesthesia at 2 days after the administration. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated by injection of Cd were significantly higher in the MT-I/II null mice than in the wild-type mice. In the MT-III null mice, ALT and AST activities were not elevated following the injection of Cd. Further, marked morphological changes such as necrosis of hepatocytes, severe hemorrhage and congestion were observed by injection of Cd in both MT-I/II null mice and wild-type mice, whereas the degree of injury was found to be more extensive in MT-I/II null mice. In contrast, only occasional damage was observed in the liver of MT-III null mice treated with the same dose of Cd. These morphological observations were consistent with the results of ALT and AST activities. In the present study, it was clearly found that MT-III null mice were resistant to Cd hepatotoxicity, although MT-I/II null mice were sensitive to its toxicity. MT-III may be an accelerative factor in Cd-induced acute hepatotoxicity.
To develop an analytical method for methylmercury (MeHg) compounds in biological samples (hair, blood, fish) with reproducibly high recovery of MeHg using heating vaporization atomic absorption spectrometry, we examined the pretreatment process of biological samples such as solubilization, degreasing and solvent extraction of MeHg using the Magos method. Samples were solubilized with NaOH at 70°C and degreased of fat components by chloroform and hexane. Hydrobromic acid (HBr) was used to acidify the solubilized solution to form MeHgBr with high solvent transferability. The fraction containing MeHg-L-cysteine complexes in aqueous solution, which had been reverse-extracted from the toluene layer, were determined as MeHg. Recoveries of MeHgBr were ≈ 95% with little variation. Analytical values for standards materials for hair and fish using the present method agreed well with certified values.
The purpose of this study was to examine whether intracellular metallothionein (MT) protects against benzo[a]pyrene (B[a]P)-induced forestomach and lung carcinogenesis. Ten-week-old male MT-I/II null mice and wild-type mice were orally administered B[a]P at a dose of 100 or 250 mg/kg twice a week for 4 weeks. B[a]P-induced mortality was higher in the MT-I/II null mice than in the wild-type mice. The incidence of tumors in the forestomach and lung was 78.6% and 7.1% in the wild-type mice treated with 100 mg/kg B[a]P, respectively. In the MT-I/II null mice treated with B[a]P, tumor incidence in the forestomach and lung was 100% and 33.3%, respectively. The tumor area in the forestomach and lung in the MT-I/II null mice treated with B[a]P was greater than that of wild-type mice. These results suggest that MT acts as a biological protective factor against carcinogenesis induced by B[a]P.
A recombinant whole-cell bacterial sensor for highly selective and sensitive detection of the bioavailable methylmercury in the environment was constructed. The biosensor carries luciferase gene, luxAB, from Vibrio harveyi as a reporter under the control of the mercury inducible regulatory part of mer-operon from Pseudomonas K-62 plasmid pMR26. In addition, a merB gene encoding organomercurial lyase which cleaves the C-Hg bond of methylmercury to give Hg2+ was coexpressed in the sensor. The resulting bacterial sensor responded specifically to methylmercury, and the lowest detectable concentration of methylmercury was 10 pM with 1 ml sample in the optimized assay conditions. This detection limit is enough to detect this compound in many contaminated and some pristine environmental samples.
Miniature swine (minipigs) are often used in non-rodent toxicity studies. However, unlike other animal species, the parathyroid glands of minipigs are often covered with thymic tissue and are similar in color, making macroscopical identification difficult. We investigated a method for sampling and tissue preparation of the parathyroid glands using 5- to 7-month-old minipigs. The glands were identified by finding the insertion site of a branch from the carotid artery into the cervical part of the thymus. Then the glands were marked and sampled. In a preliminary study, the glands were macroscopically and microscopically detected in 3/8 animals. The glands were not identified macroscopically in 5/8 animals but were detected in 3 of these animals. In total, we succeeded in detection of the glands in 6/8 animals (75%). The method was applied in the main toxicity study, and we succeeded in 100% detection through technical advancement. The method described herein enables high rate of detection and is useful in the pathological evaluation of the parathyroid glands of minipigs.
The androgen receptor (AR) binding assay can be used to determine the ability of probable endocrine disruptors (EDs) to compete with synthetic androgen methyltrienolone (R1881) for binding to recombinant rat AR (rrAR). In this study, we assessed AR binding of various chemicals using Lexius Freyberger’s method. The rank of relative binding affinity (RBA, IC50) on the tested chemicals was trenbolone 1.3 × 10-8 M (RBA 138) > dihydrotesterone (DHT) 1.8 × 10-8 M (RBA 100) > methyl testosterone 5.7 × 10-8 M (RBA 31.6) > nonylphenol (NP) 1.3 × 10-5 M (RBA 0.14) > bisphenol A (BPA) 1.1 × 10-4 M (RBA 0.016) > isobutyl paraben 3.1 × 10-4 M (RBA 0.0058) > butyl paraben 6.2 × 10-4 M (RBA 0.0029) > propyl paraben 9.7 × 10-4 M (RBA 0.0019). However, di(n-butyl) phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP), known anti-androgenic chemicals, did not show any significant AR binding activity. Our data suggests that in vitro AR binding assay may be useful as a screening tool for potential EDs.
Tributyltin chloride (TBT) is a neurotoxic environmental pollutant that inhibits mitochondrial adenosine triphosphate (ATP) synthase. Autophagy is one of the major protein degradation systems induced by a decrease of intracellular ATP following activation of AMP-activated protein kinase (AMPK). Because we previously found that TBT induces activation of AMPK, here we examined whether TBT induces autophagic neuronal death. Exposure of cortical neurons to 500 nM TBT reduced the phosphorylation of mammalian target of rapamycin (mTOR), a regulator of autophagy. An autophagy inhibitor, 3-methyladenine (3-MA), markedly decreased TBT-induced neuronal death. TBT also induced the formation of LC3-II, an autophagy marker. These results suggest that TBT-induced neuronal death is at least partly autophagic.
Quantitative analysis of metabolites is important in 1H-nuclear magnetic resonance (NMR)-based metabolomics of plasma. Human plasma contains a high density of proteins which heavily adsorb the commonly-used standard compound of sodium 3-(trimethylsilyl) propionate 2, 2, 3, 3-d4 (TSP). We have evaluated calcium formate as an alternative standard in 1D single-pulse 1H-NMR spectra to quantify plasma metabolites. Formate did not interact with either plasma metabolites or proteins under adequate conditions. Linear relations between the signal intensities and the added formate have been demonstrated in 1H spectra. The quantifications of glucose and creatinine by this method have shown good accordance with biochemical analysis. Calcium formate is applicable as a concentration standard to NMR metabolomics of plasma.
Paraquat is one of the most widely used herbicides in the world and has been known to injure lungs, liver and skin in animals and human. Hence, it is important to understand the manner of paraquat in mammals. We studied the effect of paraquat on the immune function of mouse in vitro and in vivo. When splenocytes were cultured in vitro with various concentrations of paraquat, IgA productivity was not affected while IgG and IgM productivity decreased. On the other hand, Oral administration of paraquat for 1, 2 or 3 weeks increased IgA level but decreased IgM levels in serum of mice. Similarly IgA productivity increased while IgM productivity decreased. These results suggest that paraquat perturbs the lymphocytes immunoglobulin productivity in an immunoglobulin class-dependent manner.
Expressed in renal carcinoma (ERC)/mesothelin is a good biomarker for human mesothelioma and has been investigated for its mechanistic rationale during the mesothelioma development. Studies are thus ongoing in our laboratories to assess expression of ERC/mesothelin in sera and normal/proliferative/neoplastic mesothelial tissues of animals untreated or given potentially mesothelioma-inducible xenobiotics, by an enzyme-linked immunosorbent assay (ELISA) for N- and C-(terminal fragments of) ERC/mesothelin and immunohistochemistry for C-ERC/mesothelin. In the present paper, we intend to communicate our preliminary data, because this is the first report to show how and from what stage the ERC/mesothelin expression changes during the chemical induction of mesothelial proliferative/neoplatic lesions. Serum N-ERC/mesothelin levels were 51.4 ± 5.6 ng/ml in control male Fischer 344 rats, increased to 83.6 ± 11.2 ng/ml in rats given a single intrascrotal administration of 1 mg/kg body weight of multi-wall carbon nanotube (MWCNT) and bearing mesothelial hyperplasia 52 weeks thereafter, and further elevated to 180 ± 77 ng/ml in rats similarly treated and becoming moribund 40 weeks thereafter, or killed as scheduled at the end of week 52, bearing mesothelioma. While C-ERC/mesothelin was expressed in normal and hyperplastic mesothelia, the protein was detected only in epithelioid mesothelioma cells at the most superficial layer. It is thus suggested that ERC/mesothelin can be used as a biomarker of mesothelial proliferative lesions also in animals, and that the increase of levels may start from the early stage and be enhanced by the progression of the mesothelioma development.
In order to elucidate the effect of metallothionein (MT)-III on hepatic gene expression altered by cadmium (Cd), we examined gene expression patterns in the liver of MT-III null mice and wild-type mice after Cd injection using a DNA microarray containing 35,852 genes. In a comparison between Cd-injected MT-III null mice and Cd-injected wild-type mice, 9 genes were found to be up-regulated and 28 genes—including serum amyloid A1 (SAA-1) and SAA-2—were down-regulated.
DNA microarray was used to monitor the transcriptional response of human brain microvascular endothelial cells (HBMEC) and human coronary artery endothelial cells (HCAEC) to sodium arsenite (iAsIII). iAsIII enhanced the expression of 10 and 6 genes, and reduced the expression of 45 and 20 genes in HBMEC and HCAEC, respectively. Among the 64 genes whose expression was changed in some way, HBMEC and HCAEC had 5 up-regulated and 12 down-regulated genes in common.