Assessment of oxidative stress responses and the cytotoxic and genotoxic potential of the herbicide tembotrione in HepG2 cells

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus “cytome” assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.     

Strawberry consumption alleviates doxorubicin-induced toxicity by suppressing oxidative stress

Doxorubicin (Dox), one of the most used chemotherapeutic agents, is known to generate oxidative stress and block DNA synthesis, which result in severe dose-limiting toxicity. A strategy to protect against Dox toxic effects could be to use dietary antioxidants of which fruits and vegetable are a rich source. In this context, strawberry consumption is associated with the maintenance of good health and the prevention of several diseases, thanks to the antioxidant capacities of its bioactive compounds. The aim of the present study was to evaluate the protective effects of strawberry consumption against oxidative stress induced by Dox in rats. Animals were fed with strawberry enriched diet (15% of the total calories) for two months and Dox (10 mg/kg; i.p.) was injected at the end of the experimental period. Strawberry consumption significantly inhibited ROS production and oxidative damage biomarkers accumulation in plasma and liver tissue and alleviated histopathological changes in rat livers treated with Dox. The reduction of antioxidant enzyme activities was significantly mitigated after strawberry consumption. In addition, strawberry enriched diet ameliorated liver mitochondrial antioxidant levels and functionality. In conclusion, strawberry intake protects against Dox-induced toxicity, at plasma, liver and mitochondrial levels thanks to its high contents of bioactive compounds.     

Atorvastatin induced hepatic oxidative stress and apoptotic damage via MAPKs,mitochondria, calpain and caspase12 dependent pathways

Atorvastatin (ATO), a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, is used widely for the treatment of hypercholesterolemia and hypertriglyceridemia. Application of this drug has now been made somehow limited because of ATO associated several acute and chronic side effects. The present study has been carried out to investigate the dose-dependent hepatic tissue toxicity in ATO induced oxidative impairment and cell death in mice. Administration of ATO enhanced ALT, ALP level, increased reactive oxygen species (ROS) production and altered the pro oxidant-antioxidant status of liver by reducing intracellular GSH level, anti-oxidant enzymes activities and increasing intracellular lipid peroxidation. Our experimental evidence suggests that ATO markedly decreased mitochondrial membrane potential, disturbed the Bcl-2 family protein balance, enhanced cytochrome c release in the cytosol, increased the levels of Apaf1, caspase-9, -3, cleaved PARP protein and ultimately led to apoptotic cell death. Besides, ATO distinctly increased the phosphorylation of p38, JNK, and ERK MAPKs, enhanced Caspase12 and calpain level. Histological studies also support the dose-dependent toxic effect of ATO in these organs pathophysiology. These results reveal that ATO induces hepatic tissue toxicity via MAPKs, mitochondria and ER dependent signaling pathway, in which calcium ions and ROS act as the pivotal mediators of the apoptotic signaling.     

Toxicity and oxidative stress induced by T-2 toxin and HT-2 toxin in broilers and broiler hepatocytes

T-2 and HT-2 toxins belong to mycotoxins which are found in human foods and animal chow. We investigated the toxicity and oxidative stress induced by T-2/HT-2 in broilers and chicken hepatocytes. Maize cultures of Fusarium poae was fed to broilers for 42 d, and the physiological index, biochemical index and expression of mRNAs related to oxidative stress were analyzed. Chicken hepatocytes were treated with different levels of T-2/HT-2, and the following parameters were detected: cell viability, GSH and MDA concentration, LDH leakage, activities of ALT/AST, ROS, GSH-PX, SOD and CAT, and expression of mRNA related to oxidative stress. In vivo, high levels of mycotoxins (4 mg/kg T-2 and 0.667 mg/kg HT-2) in feed caused significant reductions in body weight, weight gain, and serum total protein, and significant increases in feed conversion ratio, ALP, ALT/AST activities, and expression of mRNA related to oxidative stress. In vitro, cells treated with T-2/HT-2 showed reductions of GSH concentration and significant increases in LDH leakage, ALT/AST ROS, GSH-PX, SOD and CAT activities, MDA concentration, and expression of mRNA related to oxidative stress. Consequently, F. poae culture material and T-2/HT-2 induced toxicity and oxidative stress in vivo and in vitro, respectively.     

Cisplatin and resveratrol induce apoptosis and autophagy following oxidative stress in malignant mesothelioma cells

Malignant mesothelioma (MM) is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. Here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on MM cells. The combination treatment of cisplatin and resveratrol (CDDP/RSV) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of sub-G0/G1 peak, an increase in the Annexin V(+) cells and the cleavage of caspase-3 and PARP. CDDP/RSV increased ROS production and depolarization of mitochondrial membrane potential with an increase in the Bax/Bcl-2 ratio. These changes were attenuated by pretreatment with N-acetylcysteine, suggesting that CDDP/RSV induced apoptosis through oxidative mitochondrial damage. Compared with MSTO-211H cells, CDDP/RSV was less efficient in killing H-2452 cells. H-2452 cells exhibited an enhanced autophagy to CDDP/RSV, as observed by an increase in viable cells exhibiting intense LysoTracker Red staining and up-regulation of Beclin-1 and LC3A. Inhibition of autophagy by bafilomycin A1 rendered cells more sensitive to CDDP/RSV-induced cytotoxicity and this was associated with induction of apoptosis. These data indicate that the increased resistance of H-2452 cells to CDDP/RSV is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of MM.     

Feed supplemented with polyphenolic byproduct from olive mill wastewater processing improves the redox status in blood and tissues of piglets

In the present study, a polyphenolic byproduct from olive mill wastewater (OMWW) was used for making piglet feed with antioxidant activity. For examining the antioxidant capacity of the feed, 30 piglets of 20 d old were divided into two groups receiving basal or experimental feed for 30 d. Blood and tissue samples were drawn at days 2, 20, 35 and 50 post-birth. The tissues collected were brain, heart, kidney, liver, lung, quadriceps muscle, pancreas, spleen and stomach. The antioxidant effects of the experimental feed were assessed by measuring oxidative stress biomarkers in blood and tissues. The oxidative stress markers were total antioxidant capacity (TAC), glutathione (GSH), catalase activity (CAT), protein carbonyls (CARB) and thiobarbituric acid reactive species (TBARS). The results showed that piglets fed with diet supplemented with OMWW polyphenols had significantly increased antioxidant mechanisms in blood and the majority of the tested tissues as shown by increases in TAC, CAT and GSH compared to control group. Moreover, piglets fed with the experimental feed exhibited decreased oxidative stress-induced damage to lipids and proteins as shown by decreases in TBARS and CARB respectively. This is the first study in which OMWW polyphenols were used for making pig feed with antioxidant activity.     

A two-year dietary carcinogenicity study of cyadox in Sprague-Dawley rats

To investigate the potential carcinogenicity of cyadox, an antimicrobial agent, four groups of Sprague-Dawley rats (50 rats/sex/group) were fed diets containing cyadox (0, 200, 600 or 2000 mg/kg) for up to two years. There were significant decreases in body weight, feed intake and feed efficiency in both genders during most of the period in the 2000 mg/kg group. Significant decreases in serum ALT were observed in the 2000 mg/kg group at weeks 52, 78 and 104. For the control, 200, 600, and 2000 mg/kg groups, the tumor incidence in females was 33.3%, 37.2%, 40.0% and 19.0%, while it in males it was 18.9%, 2.6%, 17.1% and 13.6%, respectively. At histopathology, no increases in tumor incidence were attributed to treatment with cyadox. The mild swelling and fatty degeneration in hepatocytes, and mild swelling and tubular necrosis in the kidney were observed in 2000 mg/kg group. The no-observed-effect-level (NOEL) for carcinogenicity of cyadox fed to rats was 2000 mg/kg diet (132.18–156.28 mg/kg b.w./day). In conclusion, cyadox was not carcinogenic to rats with the liver and kidney as the target organs, and the side chain may be involved in toxicity and carcinogenicity mediated by QdNOs.     

Acute and subacute toxicity studies of CMICE-013, a novel iodinated rotenone-based myocardial perfusion tracer,in Sprague Dawley rats and Gottingen minipigs

PurposeExtensive acute and subacute toxicities studies are required to evaluate the toxicological profile of the novel cardiac perfusion imaging tracer ¹²³I-CMICE-013 to support applications for clinical trials.MethodsSprague-Dawley rats and Gottingen minipigs received injections of non-radioactive 127I-CMICE-013 at two dosage levels of 1 and 5 μg/kg, and vehicle buffer as control. In the acute toxicity studies, each animal was injected on two occasions 24 h apart and then underwent a 14-day recovery period; in the subacute study, animals received daily injections for 14 days continuously. The health status and mortality of test animals were monitored daily and body weight, food consumption, physiological and biochemical parameters were measured at various time points during the study. Animals were euthanized at the end of the studies and dissected for pathologic examination of organs and tissues.ResultsThe acute and subacute administrations of injections of the non-radioactive CMICE-013 in rats and minipigs were well tolerated. Little to no dosing-related adverse effects were observed in animal body and organ weights, hematology, coagulation, clinical chemistry, urinalysis, ophthalmoscopy, electrocardiograms, heart rates, blood pressure, macroscopic and microscopic examination of the preserved animal tissues including the brain.ConclusionThe lack of adverse effects from acute and subacute dosing suggest that the CMICE-013 injection solution has a reasonable safety margin within the designed concentration range to be utilized in imaging applications. The dosage level of 5 μg/kg was considered the no adverse effect level for both rats and minipigs based on our acute and subacute studies.     

Low-dose and combined effects of oral exposure to bisphenol A and diethylstilbestrol on the male reproductive system in adult Sprague-Dawley rats

Study of the joint action of xenobiotics is important to fully explore their toxicity and complete risk analysis. In this study, we investigated the effects of low-dose and combined exposure of bisphenol A (BPA) and diethylstilbestrol (DES) on the reproductive system in adult male rats. The results showed that the sperm motility decreased in the BPA/DES and combined groups. Sperm deformity ratios and histological lesions of the testes were significantly higher and more significant, respectively, in the combined group compared with the single treated groups. No dose-effect relationship or significant additive effect on serum hormone levels was observed after combined exposure to BPA/DES. Ultrastructural results showed lesions of the Sertoli and Leydig cells, mainly in the endoplasmic reticulum (ER), in all treated groups. ER stress molecular sensor IRE1 was phosphorylated and activated after BPA and DES treatment in this study. The protein levels of ES stress molecular marker CHOP were significantly up-regulated after exposure to BPA, DES, and BPA and DES combined. These findings indicate that ER stress is important in BPA/DES-induced damage in rat testes. Low-dose and combined exposure to BPA and DES may have toxic effects on male fertility in the adult population.     

The effect of phytosterol protects rats against 4-nitrophenol-induced liver damage

We investigated the effect of phytosterol (PS) in regard to liver damage induced by 4-nitrophenol (PNP). Twenty rats were randomly divided into four groups (Control, PS, PNP, and PNP + PS). The PS and PNP + PS groups were pretreated with PS for one week. The PNP and PNP + PS groups were injected subcutaneously with PNP for 28 days. The control group received a basal diet and was injected with vehicle alone. Treatment with PS prevented the elevation of the total bilirubin levels, as well as an increase in serum alkaline transaminase and aspartate transaminase, which are typically caused by PNP-induced liver damage. Histopathologically showed that liver damage was significantly mitigated by PS treatment. However, there was no significant change in antioxidant enzyme activities, and the Nrf2-antioxidant system was not activated after treatment with PS. These results suggest that PS could mitigate liver damage induced by PNP, but does not enhance antioxidant capacity.     

Cyto-genotoxicity and oxidative stress induced by zinc oxide nanoparticle in human lymphocyte cells in vitro and Swiss albino male mice in vivo

ZnO-np has immense potential and application in cosmetic and health care sectors. Hence it was imperative to assess the toxicity/safety of these nanoparticles. In this study, we have evaluated the effects of ZnO-np in human peripheral blood mononuclear cells (PBMCs) in vitro and in Swiss albino male mice in vivo for cyto-genotoxicity and oxidative damage. In vitro results showed that ZnO-nps were weakly genotoxic, induced significant decrease in mitochondrial membrane potential and was capable of ROS generation, leading to apoptosis. In bone marrow cells in vivo, reduction of mitochondrial membrane potential (MMP), increased oxidative stress and G0/G1 cell cycle arrest was observed along with chromosome aberrations and micronuclei formation. In liver cells DNA damage and induction of oxidative stress with concurrent decrease in inhibition of antioxidant enzymes were noted. These in vitro and in vivo results demonstrated that ZnO-np induced genotoxic response and ROS production leading to apoptotic cell death and established a good co-relation between the two biological systems. More importantly, the results stress on the need of multiple endpoint assay-approaches, with an in vitro-in vivo study design to assess nanoparticle toxicology.     

Trichloroethylene exposure aggravates behavioral abnormalities in mice that are deficient in superoxide dismutase

Trichloroethylene (TCE) has been implicated as a causative agent for Parkinson’s disease (PD). The administration of TCE to rodents induces neurotoxicity associated with dopaminergic neuron death, and evidence suggests that oxidative stress as a major player in the progression of PD. Here we report on TCE-induced behavioral abnormality in mice that are deficient in superoxide dismutase 1 (SOD1). Wild-type (WT) and SOD1-deficient (Sod1^−/−) mice were intraperitoneally administered TCE (500 mg/kg) over a period of 4 weeks. Although the TCE-administrated Sod1^−/− mice showed marked abnormal motor behavior, no significant differences were observed among the experimental groups by biochemical and histopathological analyses. However, treating mouse neuroblastoma-derived NB2a cells with TCE resulted in the down regulation of the SOD1 protein and elevated oxidative stress under conditions where SOD1 production was suppressed. Taken together, these data indicate that SOD1 plays a pivotal role in protecting motor neuron function against TCE toxicity.     

Investigation of the mechanism of triclosan induced mouse liver tumors

Chronic dietary exposure to Triclosan (TCS) produced increased incidence of liver tumors in mice. The mechanism for liver tumor induction has been attributed to activation of either peroxisome proliferator activated receptor α (PPARα) or constitutive androstane receptor (CAR). To further define the mechanism of TCS induced liver tumors, male CD-1 and C57BL/6 mice were treated with TCS at 0, 10, 100 and 200 mg/kg diet/day for 14 or 28 days. In addition, a recovery group and positive control groups for CAR or PPARα activation with either phenobarbital or diethylhexyl-phthalate were included in the 14-day study. TCS induced a dose-dependent increase in relative liver weight and centrilobular hypertrophy in both strains of mice. Hepatocyte DNA synthesis (BrdU labeling) was also increased in a dose-related pattern. In comparison with previous studies, TCS induced a significant increase in CAR/PXR (Cyp2b10, Cyp3a11) and PPARα (Cyp4a10) responsive genes in both CD-1 and C57BL/6 mice. The corresponding enzyme activity for CAR (7-pentoxyresorufin-O-dealkylase) and PPARα (peroxisomal Acyl-CoA oxidase) were also significantly increased in a similar fashion. Oxidative stress related genes Gpx1 and Aox1 were increased in the C57BL/6 but not in CD-1 mice. The increases in gene expression and enzyme activities returned to control levels after 14-day recovery. The present results demonstrate that both CAR and PPARα activation are involved in the TCS induced mouse liver tumor.     

Acute toxicity, 28-day repeated-dose toxicity and toxicokinetic study of timosaponin BII in rats

Timosaponin BII (TBII), a major steroidal saponin isolated from Anemarrhena asphodeloides Bge., displays a variety of promising pharmacological activities, such as neuroprotection, enhancement of learning and memory, vascular protection and inhibition of platelet aggregation; therefore, it has been developed as a pharmaceutical for prevention or treatment of dementia. Given the safety concerns surrounding timosaponins and the absence of studies on the safety of TBII, the potential toxicity of TBII was evaluated in toxicity and toxicokinetic studies in rats. In the acute oral toxicity study, loose stools were observed in rats receiving 4000 mg/kg, and the symptoms recovered within 1 day. In the 28-day repeated-dose oral toxicity and toxicokinetic study, rats receiving 540 mg/kg showed loose stools and a slight deceleration of body weight growth in both sexes, and the females also showed a slight decrease in food consumption. Moreover, urinalysis indicated reversible treatment-related toxicity in rats receiving 540 mg/kg. The toxicokinetic study demonstrated a dose-dependent increase in systematic exposure to TBII after 28 successive days of oral treatment with TBII. The accumulation coefficients of TBII were 4.35, 1.70 and 1.81, respectively, in rats that received 60, 180 and 540 mg/kg. The no-observed-adverse-effect level (NOAEL) is proposed to be 180 mg/kg.     

A 28-day oral toxicity study of echimidine and lasiocarpine in Wistar rats

Pyrrolizidine alkaloids (PAs) are a class of naturally-occurring plant toxins. Echimidine is one of the predominant PAs found in honeys produced in Australia and New Zealand. There is a lack of information on the oral toxicity of echimidine on which to base regulatory decisions concerning the risk to humans of these honeys. This GLP study was conducted to assess the subchronic dietary toxicity of echimidine to rats compared to that of lasiocarpine as a positive control. Wistar rats, 10/sex, were fed diets containing 0, 0.6, 1.2 or 2.5 mg/kg bw echimidine. Positive control groups, 10/sex, were fed diets containing 0.6, 1.2 or 2.5 mg/kg bw lasiocarpine.Neither PA had any effect on survival, food consumption, clinical signs, gross lesions, or histopathology. Consumption of lasiocarpine, but not echimidine, decreased bodyweight gain in males at ≥ 1.2 mg/kg bw, and in females at 2.5 mg/kg bw. Slight alterations in white cell counts and serum ALT concentrations at 2.5 mg/kg bw of both PAs were not clinically significant, had no histological correlates, and were considered to be of equivocal relevance.In conclusion, the subchronic No Observed Adverse Effect Level (NOAEL) for echimidine is 2.5 mg/kg bw/day, whereas, on the basis of a treatment-related decrease in bodyweight gain in males at 1.2 mg/kg bodyweight, the NOAEL for lasiocarpine is 0.6 mg/kg bw/day.     

A subchronic toxicity study of Radix Dipsaci water extract by oral administration in F344 rats

Radix Dipsaci, the dried root of Dipsacus asperoides C.Y. Cheng & T.M.Ai, has therapeutic effects on various disorders, and in particular, bone and joint disease. Despite such ethnomedicinal benefits, there is very little information regarding its in vivo toxicity or adverse effects. This study was conducted to evaluate the potential toxicity of the Radix Dipsaci water Extract (RD-wE) by using F344 rats. The RD-wE was administered orally to rats at doses of 0, 125, 250, 500, 1000, and 2000 mg/kg body weight (bw)/day for 13 weeks. During the treatment period there were no mortalities attributed to RD-wE. Moreover, no toxic effects were observed with regard to body weight, clinical pathology (hematology, clinical biochemistry, and urinalysis), and anatomic pathology (gross findings, organ weight, and microscopic examination). The changes related to the treatment were excessive salivation at the mouth and soft feces, observed in male and female rats at 1000 or 2000 mg/kg bw/day, but these were not accompanied by any microscopic correlate or other pathophysiological changes. Based on these results, the oral no-observed-adverse-effect level of the RD-wE was considered to be 2000 mg/kg bw/day in both genders, although the target organs were not determined under the current experimental conditions.