Recently, troponin T (TnT) and troponin I (TnI) have been reported as suitable biomarkers of myocardial injury for pre-clinical toxicity studies. The purpose of the present study was to investigate the characteristics of troponins as myocardial damage biomarkers in cynomolgus monkeys. Initially, tissue distribution of biomarkers was investigated in nine organs (including the heart, liver, and kidneys) collected from naive cynomolgus monkeys. The results showed that TnT and TnI were distributed specifically in the heart, and were not detected in other tissues. Secondly, changes in blood biomarker levels and histopathological changes in cardiac tissue were investigated following myocardial injury induced by concomitant administration of isoproterenol (ISO) and vasopressin (VASO). Compared with pre-dosing, TnT and TnI were markedly increased in the ISO + VASO groups, in which severe histopathological changes including necrosis and vacuolation of muscle fibers were observed. In order to investigate the relationship of biomarker levels with the severity of myocardial injury, Spearman's correlation coefficient was calculated between Cmax and AUC and necrosis and vacuolation scores in the heart. A high correlation between necrosis and vacuolation in the heart and TnT and TnI levels was noted. These results suggest that TnT and TnI possess high sensitivity and specificity for myocardial injury in cynomolgus monkeys, and are useful biomarkers for detection of drug-induced myocardial injury in cynomolgus monkeys.
Effects of repeated administration of phenobarbital (PB) on blood coagulation-related parameters were examined in non-pregnant, pregnant and lactating rats, and also in pups born to PB-treated lactating dams. PB was orally administered at a dose level of 80 mg/kg/day to pregnant (from gestation day (GD) 13), postpartum (from postpartum day (PPD) 7) and non-pregnant rats (from 13 weeks of age) for 7 days. Blood was collected on GD20 or PPD14 to perform blood coagulation examination. Concurrently, the blood coagulation parameters were examined in the pups. Increases in liver weight and/or hepatic cytochrome P450 content were observed in the PB-treated non-pregnant, pregnant and lactating rats. Activated partial thromboplastin time (APTT) was prolonged and anti-thrombin III (ATIII) concentration was increased in the lactating rats, while there were no changes in prothrombin time (PT) or APTT in the non-pregnant and pregnant rats. Moreover, prolongation of PT and APTT and decreases in factors VII and IX activities were observed in their pups. Thus, prolongation of blood coagulation time was confirmed in both dams and their pups following PB-administration to lactating dams. Effects of vitamin K2 (VK2) on PB-induced changes in blood coagulation-related parameters of both dams and their pups were examined by co-administration with PB and VK2 to lactating dams. PT and APTT were comparable to the control and PB-induced prolongation of blood coagulation time was improved in the pups while APTT was prolonged in dams, suggesting that VK2 was beneficial to pups but not to dams.
Short time exposure (STE) test using rabbit corneal cell line (SIRC) cells was developed as an alternative eye irritation test. STE test uses relative viability as the endpoint after cells are exposed to the test material at constant concentrations for 5 min. In this inter-laboratory study with 3 laboratories, 44 chemicals with a wide range of classes were evaluated for the transferability, between-lab reproducibility and predictive capacity of the STE test as an alternative eye irritation test. Globally harmonized system (GHS) classification based on Draize eye irritation test data was used as the comparative in vivo data. Transferability was assessed using standard chemicals (sodium lauryl sulfate, calcium thioglycolate, and Tween 80) and the coefficient variations (CVs) of relative viabilities between 3 labs were less than 0.13. The irritation category (Irritant or Non irritant) at each test concentration (5% and 0.05%) in STE test was the same in 3 laboratories for all 44 tested chemicals. The predictive capacity irritation category classification between STE test and GHS were compared, and a good correlation was confirmed (accuracy was 90.9% at all laboratories). In addition, the STE rankings of 1, 2, and 3 classified by the prediction model (PM) based on the relative viability at two concentrations (5% and 0.05%) were highly correlated with the GHS ranks of non-irritant, category 1, and category 2, respectively (accuracy was 75.0% at all laboratories). These results suggest that the STE test possessed easy transferability, reproducibility, good predictive performance.
The catechol metabolites (2-OHE and 4-OHE) of estrogen enter a redox cycle, thereby generating not only reactive oxygen species (ROS) but also electrophilic quinones. It is well recognized that chemicals causing oxidative stress or electrophiles activate a transcription factor Nrf2 that is negatively regulated by Keap1, leading to up-regulation of downstream proteins responsible for detoxification of electrophiles in cells. The purpose of the present study is to explore the roles of oxidative and electrophilic stress in Nrf2 activation caused by redox-active catechol estrogens. Exposure of RAW264.7 cells to 2- and 4-OHE activated Nrf2, resulting in induction of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC). Under these conditions, intracellular oxidants were generated; however, subsequent examinations revealed that quinoid metabolites derived from 2- and 4-OHE mainly participate in the Nrf2 activation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis revealed Keap1 undergoes modification by such quinoid species through multiple reactive thiol groups. These results suggest that Nrf2 activation during redox cycling of catechol estrogens is dominantly attributable to formation of their ortho-quinones that covalently bind to Keap1.
The aim of this study was to evaluate the effect of green tea in inhibiting and reversing the nephrotoxicity of reserpine - a potent oxidative stress inducer - which induced cellular kidney damage. Serum biochemical parameters, antioxidant enzyme levels, thiobarbituric acid reactive substances (TBARS) and serum transaminases (glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT)) values and histopathology were systematically evaluated. Reserpine exposure led to increase the oxidative stress and organ injury was significantly observed through biochemical parameters and ultrastructural evaluation. Sprague-Dawely (S.D.) rats were intraperitonealy administered reserpine to induce oxidative kidney damage. Experimental rats were given green tea extract according to the protocol given below. Sixty rats were randomly divided into six groups, with 10 rats in each group. Reserpine was found to cause kidney proximal tubule damage, such as stripping and clustering of ribosomes from the rough endoplasmic reticulum (rER) and demolishing of mitochondrial christae with elevated level of oxidative stress markers, such as TBARS. While the ultrastructural study showed a revival of kidney proximal tubule cells as a result of the administration of green tea extract to rats. We suggest that green tea might elevate antioxidant defense system, clean up free radicals, lessen oxidative damages and protect kidney against reserpine - induced toxicity and thus had a potential protective effect.
In order to characterize the hepatic effects of phenobarbital (PB) and clofibrate (CPIB) in dogs, PB and CPIB were administered to male beagle dogs for 14 days, and biochemical and histopathological examinations and comprehensive genomic and proteomic analyses, including GeneChip® analysis and proteomics analysis using the 2-dimension difference gel electrophoresis (2D-DIGE) technique, were performed. Both compounds caused centrilobular hepatocellular hypertrophy, which were related to smooth endoplasmic reticulum (SER) proliferation in PB-treated dogs and to mitochondrial proliferation in CPIB-treated dogs. In the PB-treated dogs, drug-metabolizing enzyme induction was observed by Western blot and genomic analyses. CYP proteins could not be detected by the 2D-DIGE analysis, but increases in several endoplasmic reticulum (ER)-related proteins were observed. In the CPIB-treated dogs, drug-metabolizing enzyme induction was not clearly observed by any of Western blot, genomic and proteomic analyses. Genomic and proteomic analyses revealed that mitochondrial genes and proteins, including carnitine palmytoiltransferase II, acyl-CoA deheydrogenase and hydroxyacyl-CoA dehydrogenase, pyruvate carboxylase and ATP synthase beta chain were induced. There is a relatively good correlation among the morphology and the genomic and proteomic data, but some differences exist between the genomic and proteomic data. Comprehensive evaluation using these techniques in addition to morphological evaluation may provide a useful tool for safety assessment of the liver.
Deltamethrin (DM) is a α- cyano pyrethroid insecticide used extensively in pest control. Although initially thought to be least toxic, a number of recent reports showed its toxicity in mammalian and non-mammalian laboratory and wildlife animal species. The present study, carried out in male rats, is a contribution to explore some mechanisms underlying DM toxicity. The aim of the present research was to investigate the effect of different subcutaneous treatments with DM (2 ppm for 30 days, 20 ppm for 45 days and 200 ppm for 60 days) on testes histopathology, sex hormones and oxidative stress from male rats. Our study mentioned an hypospermatogenesis within the testes accompanied by some apoptotic figures in particular cell fragments into the seminiferous tubules (ST)’ lumen. The results obtained show that follicule-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone can be differently influenced in rats. In fact, findings of the present investigation mention a significant decrease (p ≤ 0.05) of FSH, LH and testosterone at the highest DM dose. Whereas a significant reduction of FSH was noticed after 45 days of treatment. The assessment of oxidative stress by malondialdehyde (MDA) measurements in plasma revealed a significant increase of this parameter after 30 days, 45 days or 60 days. In conclusion the study shows that subcutaneous DM treatment produces an arrest of spermatogenesis, a significant disharmony in sex hormones and MDA levels in rats that is related to dose, length of treatment and to the lipid peroxidation which may be one of the molecular mechanisms involved in DM-induced gonads toxicity.
Rats exposed to the quaternary herbicide paraquat (PQ) exhibit oxidative stress and lung injury. In the present study, we investigated the effect of multiple exposures to PQ on aerobic performance during progressive exercise on a treadmill in rats. PQ was dissolved in saline (SAL) (10 mg/ml) and administered intraperitoneally 7 mg/kg body wt to Wistar rats (n = 5) once a week for one month. Control rats received SAL (0.7 ml/kg body wt., intraperitoneally, n = 5) over the same time period. The animals were submitted to aerobic evaluation on a treadmill using a progressive protocol until fatigue prior to the administration of the first dose of PQ or SAL and repeated at 1 week and 40 days following the last dose of the herbicide. Twenty-four hours after the last performance tests, the animals were sacrificed, lungs removed and divided in two groups: PQ and SAL for histopathological analysis. The animals exposed to PQ exhibited decrease in aerobic performance and mechanical efficiency (ME) as well as increase in oxygen consumption during exercise in comparison to the controls forty days after the last dose of PQ. Lung histologic changes included atelectasis, interstitial edema, and inflammation cells in PQ group. The collagen system fibers, fraction area of alveolar collapse and influx of polymorphonuclear (PMN) cells in lung parenchyma were higher in PQ compared to SAL. In conclusion, multiple exposures to PQ induce pulmonary fibrosis, reduce the aerobic performance and mechanical efficiency and increase the metabolic cost of exercise in rats.
Proteomic analysis of developmental toxicity by two-dimensional electrophoresis (2-DE) may detect gender-related toxic effects in embryos without visible gender characteristics. In the present study, we explored sexing of rat embryo stored in frozen 2-DE samples by polymerase chain reaction (PCR) of a male-specific gene sequence, sex determining region Y (Sry). The embryo proper and yolk sac membrane at gestation day 11 from Wistar rats were used for stored embryonic 2-DE samples. The embryonic 2-DE samples were desalted and their total DNA was extracted. The Sry sequence in the extracted DNA was amplified by PCR and the product was analyzed by agarose gel electrophoresis. The embryos with the PCR product of Sry were determined as male, and those without the product were determined as female. It was concluded that stored embryonic 2-DE samples could be used for retrospective examination of gender-related effects in proteomic analysis of developmental toxicity.
Recent studies showed that perfluorooctane sulfonate (PFOS) affects the mammalian immune system at levels reportedly found in the general human population. It has been demonstrated that exposure to immunotoxic chemicals may diminish the host resistance of animals to various pathogenic challenges and enhance mortality. Therefore, the current study was carried out to characterize the effect of a 21 day pre-administration of zero, 5, or 25 μg PFOS/kg bw/day in female B6C3F1 mice on host resistance to influenza A virus infection. At the end of PFOS exposure, body/organ weights did not significantly change whereas PFOS distribution in blood plasma, spleen, thymus and lung was dose-dependently increased. PFOS exposure in mice resulted a significant increase in emaciation and mortality in response to influenza A virus. The effective plasma concentrations in female mice were at least several fold lower than reported mean blood PFOS levels from occupationally exposed humans, and fell in the upper range of blood concentrations of PFOS in the normal human population and in a wide range of wild animals. Hence, it should be important to clarify the precise mechanism(s) for excess mortality observed in the high dose group.
Mutagenicity of fucoxanthinol (FXOH), the major compound after oral ingestion of fucoxanthin (FX), was evaluated by in vitro Ames test, and of FX by in vivo micronucleus test. In in vitro Ames test, bacterial reverse mutation was examined by using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and Escherichia coli WP2uvrA/pKM101, with or without metabolic activation by S9 mix in the preincubation method, and mutagenicity of FXOH was found to be negative in all cases. In in vivo micronucleus test, mice were orally administered with FX at doses of 500, 1,000 and 2,000 mg/kg, and the bone marrow cells were taken 24 hr after the administration to observe the incidence of micronucleus cells, and mutagenicity of FX was found to be negative at all doses. Based on the data of the present study it can be presumed that orally administered FX is a safe compound in terms of mutagenicity under the experimental conditions employed here.
Carbonated soft drinks reportedly contain methylglyoxal (MG), which is strongly associated with human carbonyl stress. We sought to evaluate the effects of carbonated drink intake on human carbonyl stress. We measured MG levels in 4 commercial beverage brands, and evaluated the changes in plasma MG in healthy subjects following the intake of carbonated drinks. By 30 min after intake of samples containing high glucose and high MG, the levels of plasma MG, glucose, insulin and uric acid had increased significantly, and then returned to basal levels by 120 min. After intake of the low-calorie carbonated samples containing little MG, there were no increases in plasma MG. Our results suggest that glucose-containing carbonated soft drinks are associated with increases in not only glucose but also carbonyl burden.
To elucidate the role ofribosomes in the manifestation of adriamycin toxicity, ribosome-bindingproteins involved in adriamycin sensitivity were identified using budding yeastas a eukaryotic model. This revealed that adriamycin toxicity was enhanced byloss of the Egd1 or Egd2 subunits of the nascent polypeptide-associated complex(NAC). NAC is a heterodimer consisting of α (Egd2) and β (Egd1 or Btt1)subunits, and is known to be involved in the translocation of nascentpolypeptides into mitochondria or endoplasmic reticulum and in transcriptionalactivation in the nucleus. Because the loss of the Btt1 subunit had no effecton adriamycin sensitivity, the NAC conformation responsible for resistance toadriamycin appears to be the Egd1/Egd2 complex. We propose that functional NACin the ribosome is involved in resistance to adriamycin toxicity.
Levels of heme oxygenase-1 (HO-1), a stress response protein, were measured to examine oxidative stress induced by several chemicals in HepG2 cells with and without S9mix using an ELISA. CdCl2, heme, and diclofenac sodium salt (diclofenac) were used as inducers of HO-1. Acetaminophen (AAP) and cyclophosphamide (CP) were used as oxidative stress inducers. Stannic mesoporphyrin (SnMP) was used as an inhibitor of HO activity. Cytotoxicity was determined, and HO-1 levels were measured in HepG2 cells exposed to chemicals other than CP with non-metabolic activation without S9mix, and to diclofenac, AAP and CP with metabolic activation with S9mix. HO-1 levels were increased by CdCl2 (7.5 μM), heme (10, 100 μM), and stannic mesoporphyrin (SnMP) (10 μM), but were not changed by AAP, and were decreased by diclofenac. HO-1 levels were increased by diclofenac (300 μM), and CP (36 μM), but were unaffected by AAP because of low sensitivity in HepG2 cells. The induction of HO-1 expression was first observed in cultured HepG2 cells treated with CP under conditions involving metabolic activation. These results showed the measurement of HO-1 protein levels in this system is useful when assessing oxidative stress as a tool for detecting drug toxicity.
Reactive oxygen species (ROS) produced by mitochondria are potentially involved in the manifestation of methylmercury toxicity. However, the molecular mechanism underlying methylmercury toxicity remains poorly understood. We examined susceptibility to methylmercury in yeast strains that each lacked one of components of the mitochondrial electron transport system. Resistance to methylmercury was exhibited only by yeast that lacked Rip1, a component of electron transport system complex III. Resistance to methylmercury in Rip1-deficient yeast was independent of the activity of electron transport system complex III. Also, ROS levels induced by methylmercury in Rip1-deficient yeast were significantly lower than in wild-type yeast. Thus, Rip1 is potentially involved in ROS production through an as-yet unknown mechanism that is independent of the activity of electron transport system complex III, thereby enhancing methylmercury toxicity.