NMR-based metabonomic approach on the toxicological effects of a Cimicifuga triterpenoid

Cimicifugae Rhizoma, a well‐known botanical dietary supplement, has been the subject of intense interest due to its potential application for alleviating menopausal symptom. Although there are clinic data that the Cimicifuga extract should have hepatotoxicity, no evidence on the main chemical components has been reported. Cimicidol‐3‐O‐β ‐d‐ xyloside (CX) is one of the main triterpenoids of the rhizome. This work studies the toxicological effects of CX after oral administration (50 mg kg^?1 per day) over a 7‐day period in female SD rats using metabonomic analyses of ¹H NMR spectra of urine, serum and liver tissue extracts. Histopathological studies of liver and analyses of blood biochemical parameter, such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen and creatinine revealed that CX had no negative impacts on liver and kidney. However, the metabolic signature of ¹H NMR‐based urinalysis of daily samples displayed an increment in the levels of taurine, trimethylamine‐N‐oxide (TMAO), betaine and acetate. Elevated serum levels of creatinine, glucose, alanine, TMAO and betaine and lower levels of lactate were observed. Metabolic profiling on aqueous soluble extracts of liver showed simultaneously increases in succinate, glycogen, choline, glycerophosphorylcholine, TMAO and betaine levels and reduction in valine, glucose and lactate levels. Nevertheless, no changes in any metabonomic level were found in lipid‐soluble extracts of liver. These findings indicate that CX has a slight toxicity in liver and kidney via disturbance of the metabolisms of energy and amino acids. The present study provides a reasonable explanation for the clinical hepatotoxicity of Cimicifuga extract. Copyright © 2011 John Wiley & Sons, Ltd.     

Gas chromatography–mass spectrometry‐based profiling of serum fatty acids in acetaminophen‐induced liver injured rats

In this study, we have developed and validated a simple, accurate and sensitive gas chromatography–mass spectrometry (GC‐MS) method for simultaneous quantification of 18 fatty acids in rat serum, including both non‐esterified (NEFA) and esterified (EFA) fatty acids, and subsequent analysis of fatty acid metabolic profiles. This novel method was used to evaluate the serum levels of fatty acids from vehicle‐ and acetaminophen (APAP)‐treated rats. Serum levels of 7 NEFAs and 14 EFAs were significantly higher in APAP‐treated rats 24 h after APAP administration at 1500 mg kg^–1 when compared with vehicle‐treated controls. Control and APAP‐treated rats could be differentiated based on their metabolic profiles using two different chemometric analysis methods: principle component analysis (PCA) and partial least squares‐discriminant analysis (PLS‐DA). More importantly, we identified the following NEFAs as potential biomarkers of APAP‐induced liver injury: oleic acid (C18:1n9), linoleic acid (C18:2n6), docosahexaenoic acid (C22:6n3) and arachidonic acid (C20:4n6). The serum concentrations of C18:1n9, C18:2n6 and C22:6n3 were all positively correlated (r > 0.8; Pearson's correlation analysis) with the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). These results suggest that a novel targeted metabolomics method based on the metabolic profiling of fatty acids analyzed by GC‐MS provides exact serum concentrations of fatty acids as well as a prospective methodology to evaluate chemically induced hepatotoxicity. Copyright © 2012 John Wiley & Sons, Ltd.     

Nuclear magnetic resonance‐ and mass spectrometry‐based metabolomics to study maleic acid toxicity from repeated dose exposure in rats

Maleic acid (MA), a chemical intermediate used in many consumer and industrial products, was intentionally adulterated in a variety of starch‐based foods and instigated food safety incidents in Asia. We aim to elucidate possible mechanisms of MA toxicity after repeated exposure by (1) determining the changes of metabolic profile using ¹H nuclear magnetic resonance spectroscopy and multivariate analysis, and (2) investigating the occurrence of oxidative stress using liquid chromatography tandem mass spectrometry by using Sprague–Dawley rat urine samples. Adult male rats were subjected to a 28 day subchronic study (0, 6, 20 and 60 mg kg⁻¹) via oral gavage. Urine was collected twice a day on days 0, 7, 14, 21 and 28; organs underwent histopathological examination. Changes in body weight and relative kidney weights in medium‐ and high‐dose groups were significantly different compared to controls. Morphological alterations were evident in the kidneys and liver. Metabolomic results demonstrated that MA exposure increases the urinary concentrations of 8‐hydroxy‐2′‐deoxyguanosine, 8‐nitroguanine and 8‐iso‐prostaglandin F_2α; levels of acetoacetate, hippurate, alanine and acetate demonstrated time‐ and dose‐dependent variations in the treatment groups. Findings suggest that MA consumption escalates oxidative damage, membrane lipid destruction and disrupt energy metabolism. These aforementioned changes in biomarkers and endogenous metabolites elucidate and assist in characterizing the possible mechanisms by which MA induces nephro‐ and hepatotoxicity.     

Involvement of Th2 cytokines in the mouse model of flutamide-induced acute liver injury

Drug‐induced liver injury is a growing concern for pharmaceutical companies and patients because numerous drugs have been linked to hepatotoxicity and it is the most common reason for a drug to be withdrawn. Flutamide rarely causes liver dysfunction in humans, and immune allergic reactions have been suggested in some cases. In this study, we investigated the mechanisms of flutamide‐induced liver injury in BALB/c mice. Plasma alanine aminotransferase and aspartate aminotransferase levels were significantly increased 3, 6 and 9 h after flutamide (1500 mg kg^?1, p.o.) administration. The biomarker for oxidative stress was not changed, but Th2‐dominant immune‐related factors, such as interleukin (IL)‐4, IL‐5, STAT6 and GATA‐binding protein (GATA)‐3, were induced in flutamide‐administered mice. The pre‐administration of monoclonal‐IL‐4 antibody suppressed the hepatotoxicity of flutamide. In addition, we investigated the effect of 13,14‐dihydro‐15‐keto‐PGD₂ (DK‐PGD₂; 10 µg per mouse, i.p.) administration on flutamide‐induced acute liver injury. Coadministration of DK‐PGD₂ and flutamide resulted in a significant increase in alanine aminotransferase and a remarkable increase of macrophage inflammatory protein‐2. In conclusion, we demonstrated that flutamide‐induced acute liver injury is mediated by Th2‐dominant immune responses in mice. Copyright © 2011 John Wiley & Sons, Ltd.     

GC‐MS‐based metabolomics reveals mechanism of action for hydrazine induced hepatotoxicity in rats

Gas chromatography–mass spectrometry (GC‐MS) has great advantages for analyzing organic/amino acids, which are often targets in efficacy and/or toxicity studies. Although GC‐MS has been used for the detection of many metabolic disorders, applications of GC‐MS‐based metabolomics in pharmacology/toxicology are relatively underdeveloped. We intended to investigate applicability of a GC‐MS‐based metabolomics approach for toxicological evaluation, and tried to elucidate the mechanism of hydrazine‐induced hepatotoxicity. Rats were administered hydrazine chloride orally (120 and 240 mg kg^?1), and urine, plasma and liver samples were collected at 24 or 48 h post‐dosing. Conventional clinical chemistry and liver histopathology were performed, urine and plasma were analyzed by GC‐MS, and metabolic profiles were assessed using chemometric techniques. Principal component analysis score plots showed clear separation of the groups, indicating dose‐dependent toxicity and recovery. The mechanism of toxicity was investigated based on semi‐quantification data of identified metabolites. Amino acid precursors of glutathione (cystein, glutamate and glycine) and a product of glutathione metabolism (5‐oxoproline) were elevated dose‐dependently, accompanied with elevation of ascorbate levels. In addition, intermediates of the TCA cycle were decreased, whereas participants of the urea cycle and other amino acids were increased. These alterations were associated with histopathological changes such as fatty degeneration and glycogen accumulation. Application of GC‐MS‐based metabolomics revealed that oxidative stress and GSH consumption play important roles in the etiology of hydrazine‐induced hepatotoxicity, demonstrating that this approach is a useful tool in pharmacology and toxicology for screening, elucidating mode of action and biomarker discovery. Copyright © 2010 John Wiley & Sons, Ltd.     

Hepatotoxicity of cantharidin is associated with the altered bile acid metabolism

Cantharidin (CTD) is an effective antitumor agent. However, it exhibits significant hepatotoxicity, the mechanism of which remains unclear. In this study, biochemical and histopathological analyses complemented with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS)-based targeted metabolomic analysis of bile acids (BAs) were employed to investigate CTD-induced hepatotoxicity in rats. Sixteen male and female Sprague–Dawley rats were randomly divided into two groups: control and CTD (1.0 mg/kg) groups. Serum and liver samples were collected after 28 days of intervention. Biochemical, histopathological, and BA metabolomic analyses were performed for all samples. Further, the key biomarkers of CTD-induced hepatotoxicity were identified via multivariate and metabolic pathway analyses. In addition, metabolite–gene–enzyme network and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to identify the signaling pathways related to CTD-induced hepatotoxicity. The results revealed significantly increased levels of biochemical indices (alanine aminotransferase, aspartate aminotransferase, and total bile acid). Histopathological analysis revealed that the hepatocytes were damaged. Further, 20 endogenous BAs were quantitated via UHPLC-MS/MS, and multivariate and metabolic pathway analyses of BAs revealed that hyocholic acid, cholic acid, and chenodeoxycholic acid were the key biomarkers of CTD-induced hepatotoxicity. Meanwhile, primary and secondary BA biosynthesis and taurine and hypotaurine metabolism were found to be associated with the mechanism by which CTD induced hepatotoxicity in rats. This study provides useful insights for research on the mechanism of CTD-induced hepatotoxicity.     

Effect of gender,dose, and time on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT)-induced hepatotoxicity in Fischer 344 rats

1.?The thiazolidinedione ring present in drugs available for type II diabetes can contribute to hepatic injury. Another thiazolidinedione ring-containing compound, 3-(3,5-dichlorophenyl)-2,4-thiazoli-dinedione (DCPT), produces liver damage in rats. Accordingly, the effects of gender, dose, and time on DCPT hepatotoxicity were therefore evaluated.

2.?Male rats were more sensitive to DCPT (0.4–1.0 mmol kg^?1 by intraperitoneal administration) as shown by increased serum alanine aminotransferase levels and altered hepatic morphology 24 h post-dosing. Effects in both genders were dose dependent. In males, DCPT (0.6 mmol kg^?1) produced elevations in alanine aminotransferases and changes in liver h after dosing that progressively worsened up to 12 h. DCPT-induced renal effects were mild.

3.?It is concluded that male rats are more susceptible to DCPT hepatotoxicity and that damage occurs rapidly. DCPT primarily affects the liver and can be a useful compound to investigate the role of the thiazolidinedione ring in hepatic injury. However, the gender dependency and rapid onset of DCPT hepatotoxicity require further investigation.     

Developmental toxic potential of di‐n‐propyl phthalate administered orally to rats

The objective of this study was to evaluate the developmental toxic potential of di‐n‐propyl phthalate (DnPP) in rats. Pregnant Sprague–Dawley rats were given DnPP at doses of 0 (olive oil), 0.5, 1 and 1.5 g kg^?1 per day, by gavage, on gestation days 6–20. Benchmark doses were calculated for the effects of DnPP on fetal weight and anogenital distance of the male fetuses. Maternal body weight gain was significantly reduced at 1.5 g kg^?1 per day, over gestation days 6–9. DnPP‐treated dams also showed a statistically significant increase in liver weight and a mild but statistically significant peroxisomal enzyme induction at 1 or 1.5 g kg^?1 per day. Male and female fetal body weights were significantly reduced at 1.5 g kg^?1 per day. There was a statistically significant decrease in the anogenital distance of the male fetuses at 1 and 1.5 g kg^?1 per day, and three males (of 75) showed malpositioned testis at the high dose. The mean percentage of fetuses per litter with cervical and thoracic rudimentary ribs was significantly increased at 1 and 1.5 g kg^?1 per day. Delayed ossification was seen at 1 g kg^?1 per day (phalanges) and 1.5 g kg^?1 per day (hyoid, sternebrae, and phalanges). No treatment‐related effects on prenatal viability or on fetal external or visceral malformations or variations were observed at any dose. Thus, there was no evidence of teratogenicity up to the high dose of 1.5 g kg^?1 per day. The no‐observed‐adverse‐effect level (NOAEL) for developmental toxicity was 0.5 g kg^?1 per day. Copyright © 2010 JohnWiley & Sons, Ltd.     

Subchronic hepatotoxicity evaluation of bromobenzene in Fischer 344 rats

Male Fischer 344 (F344) rats were exposed to bromobenzene (BB) for 5 days and 2, 4 and 13 weeks. BB was administered by gavage (corn oil vehicle) at doses of 0, 25, 100, 200, 300 and 400 mg kg^?1 per day. Endpoints evaluated included clinical observations, body weights, liver weights, serum chemistry, blood BB, gross pathology and liver histopathology. There were no BB exposure‐related clinical signs of toxicity. Mean body weight decreased by 5–10% compared with control in the 400 mg kg^?1 per day group. Liver weight increases were dose‐ and exposure time‐related and statistically significant at ≥25 mg kg^?1 per day. Incidence and severity of centrilobular cytoplasmic alteration and hepatocyte hypertrophy were related to dose and exposure time. At early time points (5 days and 2 weeks), centrilobular inflammation, including granulomatous areas, and necrotic and anisokaryocytic hepatocytes were observed in rats of the two highest BB dose groups. Blood BB concentrations increased linearly with dose and at 13 weeks ranged from 8 to 136 µg ml^?1 (25–400 mg kg^?1 per day). In conclusion, rats administered BB doses up to 400 mg kg^?1 per day for up to 13 weeks had mild liver effects. A NOAEL of 200 mg kg^?1 per day was selected based on the statistically significant incidence of hepatocyte hypertrophy at doses ≥ 400 mg kg^?1 per day. Copyright © 2012 John Wiley & Sons, Ltd.     

Dichloroacetate‐ and trichloroacetate‐induced oxidative stress in the hepatic tissues of mice after long‐term exposure

Dichoroacetate (DCA) and trichloroacetate (TCA) were found to be hepatotoxic and hepatocarcinogenic in rodents. To investigate the role of oxidative stress in the long‐term hepatotoxicity of the compounds, groups of mice were administered 7.7, 77, 154 and 410 mg kg^?1 per day, of either DCA or TCA, by gavage, for 4 weeks (4‐W) and 13 weeks (13‐W), and superoxide anion (SA), lipid peroxidation (LP) and DNA‐single strand breaks (SSBs) were determined in the hepatic tissues. Significant increases in all of the biomarkers were observed in response to the tested doses of both compounds in the two test periods, with significantly greater increases observed in the 13‐W, as compared with the 4‐W, period. Hepatomegaly was only observed with a DCA dose of 410 mg kg^?1 per day in the 13‐W treatment period, and that was associated with significant declines in the biomarkers, when compared with the immediately lower dose. With the exception of LP production in the 13‐W treatment period that was similarly induced by the two compounds, the DCA‐induced increases in all of the biomarkers were significantly greater than those of TCA. Since those biomarkers were significantly induced by the compounds' doses that were shown to be carcinogenic but at earlier periods than those demonstrating hepatotoxicity/haptocarcinogencity, they can be considered as initial events that may lead to later production of those long‐term effects. The results also suggest LP to be a more significant contributing mechanism than SA and DNA damage to the long‐term hepatotoxicity of TCA. Copyright © 2010 John Wiley & Sons, Ltd.     

Early metabolomics changes in heart and plasma during chronic doxorubicin treatment in B6C3F1 mice

The present study aimed to identify molecular markers of early stages of cardiotoxicity induced by a potent chemotherapeutic agent, doxorubicin (DOX). Male B6C3F₁ mice were dosed with 3 mg kg^?1 DOX or saline via tail vein weekly for 2, 3, 4, 6 or 8 weeks (cumulative DOX doses of 6, 9, 12, 18 or 24 mg kg^?1, respectively) and euthanized a week after the last dose. Mass spectrometry‐based and nuclear magnetic resonance spectrometry‐based metabolic profiling were employed to identify initial biomarkers of cardiotoxicity before myocardial injury and cardiac pathology, which were not noted until after the 18 and 24 mg kg^?1 cumulative doses, respectively. After a cumulative dose of 6 mg kg^?1, 18 amino acids and four biogenic amines (acetylornithine, kynurenine, putrescine and serotonin) were significantly increased in cardiac tissue; 16 amino acids and two biogenic amines (acetylornithine and hydroxyproline) were significantly altered in plasma. In addition, 16 acylcarnitines were significantly increased in plasma and five were significantly decreased in cardiac tissue compared to saline‐treated controls. Plasma lactate and succinate, involved in the Krebs cycle, were significantly altered after a cumulative dose of 6 mg kg^?1. A few metabolites remained altered at higher cumulative DOX doses, which could partly indicate a transition from injury processes at 2 weeks to repair processes with additional injury happening concurrently before myocardial injury at 8 weeks. These altered metabolic profiles in mouse heart and plasma during the initial stages of injury progression due to DOX treatment may suggest these metabolites as candidate early biomarkers of cardiotoxicity. Published 2016. This article is a U.S. Government work and is in the public domain in the USA     

Tissue factor antisense deoxyoligonucleotide prevents monocrotaline/LPS hepatotoxicity in mice

Tissue factor (TF) is a membranous glycoprotein that functions as a receptor for coagulation factor VII/VIIa and activates the coagulation system when blood vessels or tissues are damaged. TF was upregulated in our monocrotaline (MCT)/lipopolysaccharide (LPS) hepatotoxicity model. We tested the hypothesis that TF‐dependent fibrin deposition and lipid peroxidation in the form of oxidized low‐density‐lipoprotein (ox‐LDL) accumulation contribute to liver inflammation induced by MCT/LPS in mice. In the present study, we blocked TF using antisense oligodeoxynucleotides against mouse TF (TF‐ASO). TF‐ASO (5.6 mg kg^?1) was given i.v. to ND4 male mice 30 min after administration of MCT (200 mg kg^?1) p.o. followed after 3.5 h by LPS i.p. (6 mg kg^?1). Blood alanine aminotransferase (ALT), TF, ox‐LDL, platelets, hematocrit and keratinocyte‐derived chemokine (KC) levels were evaluated in different treatment groups. Fibrin deposition and ox‐LDL accumulation were also analyzed in the liver sections using immunofluorescent staining. The results showed that TF‐ASO significantly restored blood ALT, hematocrit and KC levels, distorted after MCT/LPS co‐treatment, as well as preventing the accumulation of ox‐LDL and the deposition of fibrin in the liver tissues, and thereby inhibited liver injury caused by MCT/LPS. In a separate experiment, TF‐ASO administration significantly prolonged animal survival. The current study demonstrates that TF is associated with MCT/LPS‐induced liver injury. Administration of TF‐ASO successfully prevented this type of liver injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Nanoparticulate silver increases uric acid and allantoin excretion in rats,as identified by metabolomics

Metabolomic investigation of rat urine was employed to identify mammalian metabolites affected by ionic or nanoparticulate silver. Female and male Wistar rats were administered silver nanoparticles (2.25, 4.5 or 9.0 mg kg^?1 body weight per day) or ionic silver (silver acetate, 9.0 mg silver kg^?1 bw per day) by oral gavage for 28 days. On day 18, urine was collected for 24 h and subjected to metabolomics with high performance liquid chromatography–quadropole time‐of‐flight mass spectrometry (HPLC‐QTOF‐MS)‐based separation and detection. Principal component analysis was subsequently applied to the data. Metabolomic differences in urine composition were found in female rats but not in male rats. Several metabolites were identified by the use of elemental composition calculated from the exact mass combined with searches in the Human Metabolome Database.The metabolite identities were eventually verified by co‐chromatography with authentic standards. Differences were found in uric acid and its degradation product, allantoin. Administration of nanoparticulate silver increased both metabolites, whereas ionic silver only increased allantoin. In conclusion, metabolomic investigation of rat urine showed that increased levels of uric acid and allantoin were associated with exposure to nanoparticulate silver. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of structural modifications on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced hepatotoxicity in Fischer 344 rats

Glitazones, used for type II diabetes, have been associated with liver damage in humans. A structural feature known as a 2,4‐thiazolidinedione (TZD) ring may contribute to this toxicity. TZD rings are of interest since continued human exposure via the glitazones and various prototype drugs is possible. Previously, we found that 3‐(3,5‐dichlorophenyl)‐2,4‐thiazolidinedione (DCPT) was hepatotoxic in rats. To evaluate the importance of structure on DCPT toxicity, we therefore studied two series of analogs. The TZD ring was replaced with: a mercaptoacetic acid group {[[[(3,5‐dichlorophenyl)amino]carbonyl]thio]acetic acid, DCTA}; a methylated TZD ring [3‐(3,5‐dichlorophenyl)‐5‐methyl‐2,4‐thiazolidinedione, DPMT]; and isomeric thiazolidinone rings [3‐(3,5‐dichlorophenyl)‐2‐ and 3‐(3,5‐dichlorophenyl)‐4‐thiazolidinone, 2‐DCTD and 4‐DCTD, respectively]. The following phenyl ring‐modified analogs were also tested: 3‐phenyl‐, 3‐(4‐chlorophenyl)‐, 3‐(3,5‐dimethylphenyl)‐ and 3‐[3,5‐bis(trifluoromethyl)phenyl]‐2,4‐thiazolidinedione (PTZD, CPTD, DMPT and DFMPT, respectively). Toxicity was assessed in male Fischer 344 rats 24 h after administration of the compounds. In the TZD series only DPMT produced liver damage, as evidenced by elevated serum alanine aminotransferase (ALT) activities at 0.6 and 1.0 mmol kg^?1 (298.6 ± 176.1 and 327.3 ± 102.9 Sigma‐Frankel units ml^?1, respectively) vs corn oil controls (36.0 ± 11.3) and morphological changes in liver sections. Among the phenyl analogs, hepatotoxicity was observed in rats administered PTZD, CPTD and DMPT; with ALT values of 1196.2 ± 133.6, 1622.5 ± 218.5 and 2071.9 ± 217.8, respectively (1.0 mmol kg^?1 doses). Morphological examination revealed severe hepatic necrosis in these animals. Our results suggest that hepatotoxicity of these compounds is critically dependent on the presence of a TZD ring and also the phenyl substituents. Copyright © 2011 John Wiley & Sons, Ltd.     

Acute toxic effects and gender‐related biokinetics of silver nanoparticles following an intravenous injection in mice

This study evaluated the acute toxicity and biokinetics of intravenously administered silver nanoparticles (AgNPs) in mice. Mice were exposed to different dosages of AgNPs (7.5, 30 or 120 mg kg^?1). Toxic effects were assessed via general behavior, serum biochemical parameters and histopathological observation of the mice. Biokinetics and tissue distribution of AgNPs were evaluated at a dose of 120 mg kg^?1 in both male and female mice. Inductively coupled plasma–mass spectrometry (ICP‐MS) was used to determine silver concentrations in blood and tissue samples collected at predetermined time intervals. After 2 weeks, AgNPs exerted no obvious acute toxicity in the mice. However, inflammatory reactions in lung and liver cells were induced in mice treated at the 120 mg kg^?1 dose level. The highest silver levels were observed in the spleen, followed by liver, lungs and kidneys. The elimination half‐lives and clearance of AgNPs were 15.6 h and 1.0 ml h^?1 g^?1 for male mice and 29.9 h and 0.8 ml h^?1 g^?1 for female mice. These results indicated that AgNPs could be distributed extensively to various tissues in the body, but primarily in the spleen and liver. Furthermore, there appears to be gender‐related differences in the biokinetic profiles in blood and distribution in lungs and kidneys following an intravenous injection of AgNPs. The data from this study provides information on toxicity and biodistribution of AgNPs following intravenous administration in mice, which represents the worst case scenario of toxicity among all the different administration routes, and may shed light in the future use of products containing AgNPs in humans. Copyright © 2012 John Wiley & Sons, Ltd.     

Toxicity assessment of manganese oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral exposure

In the near future, nanotechnology is envisaged for large‐scale use. Hence health and safety issues of nanoparticles (NPs) should be promptly addressed. Twenty‐eight‐day oral toxicity, genotoxicity, biochemical alterations, histopathological changes and tissue distribution of nano and microparticles (MPs) of manganese oxide (MnO₂) in Wistar rats was studied. Genotoxicity was assessed using comet, micronucleus and chromosomal aberration assays. The results demonstrated a significant increase in DNA damage in leukocytes, micronuclei and chromosomal aberrations in bone marrow cells after exposure of MnO₂‐NPs at 1000, 300 mg kg^–1 bw per day and MnO₂‐MPs at the dose of 1000 mg kg^–1 bw per day. Our findings showed acetylcholinestrase inhibition at 1000 as well as at 300 mg kg^–1 bw per day in blood and with all the doses in the brain indicating the toxicity of MnO₂‐NPs. Further, the doses significantly inhibited different ATPases in the brain P₂ fraction. Significant changes were observed in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in the liver, kidney and serum in a dose‐dependent manner. MnO₂‐MPs at 1000 mg kg^–1 bw per day were found to induce significant alterations in biochemical enzymes. A significant distribution was found in all the tissues in a dose‐dependent manner. MnO₂‐NPs showed a much higher absorptivity and tissue distribution as compared with MnO₂‐MPs. A large fraction of MnO₂‐NPs and MnO₂‐MPs was cleared by urine and feces. Histopathological analysis revealed that MnO₂‐NPs caused alterations in liver, spleen, kidney and brain. The MnO₂‐NPs induced toxicity at lower doses compared with MnO₂‐MPs. Further, this study did not display gender differences after exposure to MnO₂‐NPs and MnO₂‐MPs. Therefore, the results suggested that prolonged exposure to MnO₂ has the potential to cause genetic damage, biochemical alterations and histological changes. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabonomics evaluation of urine from rats administered with phorate under long‐term and low‐level exposure by ultra‐performance liquid chromatography‐mass spectrometry

The purpose of this study was to investigate the toxic effect of long‐term and low‐level exposure to phorate using a metabonomics approach based on ultra‐performance liquid chromatography‐mass spectrometry (UPLC‐MS). Male Wistar rats were given phorate daily in drinking water at low doses of 0.05, 0.15 or 0.45 mg kg^–1 body weight (BW) for 24 weeks consecutively. Rats in the control group were given an equivalent volume of drinking water. Compared with the control group, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), urea nitrogen (BUN) and creatinine (CR) were increased in the middle‐ and high‐dose groups whereas albumin (ALB) and cholinesterase (CHE) were decreased. Urine metabonomics profiles were analyzed by UPLC‐MS. Compared with the control group, 12 metabolites were significantly changed in phorate‐treated groups. In the negative mode, metabolite intensities of uric acid, suberic acid and citric acid were significantly decreased in the middle‐ and high‐dose groups, whereas indoxyl sulfic acid (indican) and cholic acid were increased. In the positive mode, uric acid, creatinine, kynurenic acid and xanthurenic acid were significantly decreased in the middle‐ and high‐dose groups, but 7‐methylguanine (N⁷G) was increased. In both negative and positive modes, diethylthiophosphate (DETP) was significantly increased, which was considered as a biomarker of exposure to phorate. In conclusion, long‐term and low‐level exposure to phorate can cause disturbances in energy‐related metabolism, liver and kidney function, the antioxidant system, and DNA damage. Moreover, more information can be provided on the evaluation of toxicity of phorate using metabonomics combined with clinical chemistry. Copyright © 2012 John Wiley & Sons, Ltd.     

Mass spectrometry-based metabolic profiling of rat urine associated with general toxicity induced by the multiglycoside of Tripterygium wilfordii Hook. f

We propose here a combined gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) metabolic profiling strategy to elucidate the toxicity in rats induced by orally administered multiglycosides of Tripterygium wilfordii Hook. f. (GTW) in multiple organs including the kidney, liver, and testis. Overnight 12-h urine samples were collected from Sprague-Dawley male rats exposed to GTW (100 mg/kg/day, n = 6) and healthy controls ( n = 6) at predose and at the 1st, 3rd, 6th, 10th, and 14th day postdose for both GC/MS and LC/MS analyses. The integrated urinary MS data were analyzed via multivariate statistical techniques such as principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) to identify the differential metabolites and pertinent altered biological pathways in response to the herbal toxin. The liver, kidney, and testis were also assessed using conventional histopathological examinations at the end point of the experiment. This work indicates that GTW caused a time-dependent toxic effect at a high dose as revealed by the perturbed metabolic regulatory network involving disorders in energy metabolism, elevated amino acid and choline metabolism pathways, as well as altered structure of gut flora. This integrated MS-based metabolic profiling approach has been able to capture and probe the metabolic alterations associated with the onset and progression of multiorgan toxicity induced by GTW, thereby permitting a comprehensive understanding of systemic toxicity for phytochemicals and other types of xenobiotic agents.     

Toxicity of new emerging pollutant tris‐(2,3‐dibromopropyl) isocyanurate on BALB/c mice

The emerging heterocyclic brominated flame retardant tris‐(2,3‐dibromopropyl) isocyanurate (TBC), widely used in reinforced plastics, has demonstrated toxicity to fish. However, little is known about its toxicity in rodents. This study aims to determine the effect of TBC on growth, biochemical parameters in serum, organs and related gene expression of both male and female BALB/c mice after gastro‐gavage administration of 0, 2, 10 and 50 mg kg^?1 TBC for 28 days. Results indicated that exposure to TBC had no effects on basic growth and food intake of mice, but significantly increased serum alanine aminotransferase levels in male mice. Histopathological analyses showed that focal necrosis (2, 10 and 50 mg kg^?1 TBC‐exposed groups) and ballooning degeneration (10 and 50 mg kg^?1 TBC‐exposed groups) were found in mouse liver, whereas transmission electron microscopy revealed dose‐dependent hepatocyte apoptosis, mitochondrial degeneration and endoplasmic reticulum dilation. Histopathological and ultrastructural assessments in the lung showed dose‐dependent hyperplasia of pulmonary alveolar epithelium, bronchial congestion, infiltration of inflammatory cells and mitochondrial swelling following TBC exposure. Our results also indicated that mitochondria are one of the major target cytoplasmic organelles for TBC, suggesting that damage in mitochondria is one of the pathways that led to toxic effects in the liver and lung of TBC‐treated groups. Moreover, TBC effectively activated the gene expression of p53 in mice liver. Our findings provide strong evidence that TBC induces significant toxicity in mice organs, especially in liver and lung, which play vital roles in detoxification and gas exchange, respectively. This research will contribute to characterize the toxic effects of TBC, which was introduced as one of the candidates for brominated flame retardant replacement. Copyright © 2014 John Wiley & Sons, Ltd.     

Can acetaminophen/dimethyl sulfoxide formulation prevent accidental and intentional acetaminophen hepatotoxicity?

An overdose of acetaminophen (APAP) causes liver injury in experimental animals and humans. The activation step (formation of reactive metabolite, N-acetyl-p-benzoquinone imine by cytochrome P450 system) and the consequent downstream pathway of oxidative stress, nitrosative stress, and inflammation play an important role in APAP-induced hepatotoxicity. Formulation of APAP with an inhibitor of the activation step would be ideal to prevent accidental and intentional APAP toxicity. Dimethyl sulfoxide (DMSO) is a common colorless, inexpensive solvent, and considered safe in human. We hypothesized that a less hepatotoxic APAP if co-formulated with DMSO. To test this hypothesis, C57BL/6 mice were given toxic dose of APAP (250 mg kg⁻¹, i.p.) mixed with different doses of DMSO (25, 50, 100, and 200 μl kg⁻¹). Six hours after APAP treatment, blood and lives were collected for analysis. In DMSO treated groups, there was dose-dependent decrease in markers of liver injury, alanine aminotransferase, and aspartate aminotransferase. Maximum protection was obtained with 200 μl DMSO kg⁻¹. DMSO was shown to inhibit the activation step by decreasing the rate of GSH depletion in vivo and inhibiting cytochrome P450 system in vitro. Also the levels of lipid peroxides, nitrate/nitrite, tumor necrosis factor-alpha, and interleukin 1β were decreased significantly. In conclusion, DMSO exerts its protective action by inhibiting the metabolic activation of APAP and thus alleviating the downstream, oxidative stress, nitrosative stress, and inflammation via indirect inhibition. Our findings suggest that replacing the current APAP with APAP/DMSO formulation could prevent accidental and intentional APAP toxicity.