Amos O. Abolaji

Involvement of oxidative stress in 4-vinylcyclohexene-induced toxicity in Drosophila melanogaster

4-Vinylcyclohexene (VCH) is a dimer of 1,3-butadiene produced as a by-product of pesticides, plastic, rubber, flame retardants, and tire production. Although, several studies have reported the ovotoxicity of VCH, information on a possible involvement of oxidative stress in the toxicity of this occupational chemical is scarce. Hence, this study was carried out to investigate further possible mechanisms of toxicity of VCH with a specific emphasis on oxidative stress using a Drosophila melanogaster model. D. melanogaster (both genders) of 1 to 3 days old were exposed to different concentrations of VCH (10 µM-1 mM) in the diet for 5 days. Subsequently, the survival and negative geotaxis assays and the quantification of reactive oxygen species (ROS) generation were determined. In addition, we evaluated RT-PCR expressions of selected oxidative stress and antioxidant mRNA genes (HSP27, 70, and 83, SOD, Nrf-2, MAPK2, and catalase). Furthermore, catalase, glutathione-S-transferase (GST), delta aminolevulinic acid dehydratase (δ-ALA-D), and acetylcholinesterase (AChE) activities were determined. VCH exposure impaired negative geotaxic behavior and induced the mRNA of SOD, Nrf-2, and MAPK2 genes expressions. There were increases in catalase and ROS production, as well as inhibitions of GST, δ-ALA-D, and AChE activities (P<0.05). Our results suggest that the VCH mechanism of toxicity is associated with oxidative damage, as evidenced by the alteration in the oxidative stress-antioxidant balance, and possible neurotoxic consequences due to decreased AChE activity, and impairments in negative geotaxic behavior. Thus, we conclude that D. melanogaster is a useful model for investigating the toxicity of VCH exposure, and here, we have provided further insights on the mechanism of VCH-induced toxicity.

Ovotoxicants 4-vinylcyclohexene 1,2-monoepoxide and 4-vinylcyclohexene diepoxide disrupt redox status and modify different electrophile sensitive target enzymes and genes in Drosophila melanogaster.

The compounds 4-vinylcyclohexene 1,2-monoepoxide (VCM) and 4-Vinylcyclohexene diepoxide (VCD) are the two downstream metabolites of 4-vinylcyclohexene (VCH), an ovotoxic agent in mammals. In addition, VCM and VCD may be found as by-products of VCH oxidation in the environment. Recently, we reported the involvement of oxidative stress in the toxicity of VCH in Drosophila melanogaster. However, it was not possible to determine the individual contributions of VCM and VCD in VCH toxicity. Hence, we investigated the toxicity of VCM and VCD (10–1000 µM) in flies after 5 days of exposure via the diet. Our results indicated impairments in climbing behaviour and disruptions in antioxidant balance and redox status evidenced by an increase in DCFH oxidation, decreases in total thiol content and glutathione-S-transferase (GST) activity in the flies exposed to VCM and VCD (p<0.05). These effects were accompanied by disruptions in the transcription of the genes encoding the proteins superoxide dismutase (SOD1), kelch-like erythroid-derived cap-n-collar (CNC) homology (ECH)-associated protein 1 (Keap-1), mitogen activated protein kinase 2 (MAPK-2), catalase, Cyp18a1, JAFRAC 1 (thioredoxin peroxidase 1) and thioredoxin reductase 1 (TrxR-1) (p<0.05). VCM and VCD inhibited acetylcholinesterase (AChE) and delta aminolevulinic acid dehydratase (δ-ALA D) activities in the flies (p<0.05). Indeed, here, we demonstrated that different target enzymes and genes were modified by the electrophiles VCM and VCD in the flies. Thus, D. melanogaster has provided further lessons on the toxicity of VCM and VCD which suggest that the reported toxicity of VCH may be mediated by its transformation to VCM and VCD.

A Safety Assessment of the Antimalarial Herb Artemisia annua During Pregnancy in Wistar Rats

Artemisia annua is a Chinese antimalarial herb that has been used for more than 2000 years. The maternal and foetal safety of the ethanolic leaf extract of therapeutically active Artemisia annua (EAA), with previously determined artemisinin yield of 1.098% was evaluated in Wistar rats. Twenty pregnant rats, divided into four study groups of saline treated (control), and test groups administered orally with 100, 200 and 300 mg/kg body weights of EAA, respectively, from gestation days (GD) 8 to 19. Following overnight fast, animals were sacrificed on GD 20, and maternal blood was collected to evaluate biochemical and haematological markers. Foetuses were carefully removed, weighed, and observed for any possible malformation. Biochemical and haematological studies revealed that EAA did not result in maternal hepatotoxicity, haematotoxicity, and hyperlipidemia. While litter size significantly decreased (p < 0.05) at 100 mg/kg EAA, maternal estrogen levels decreased in all the EAA‐treated groups. Non‐viable (21%) and malformed (31%) foetuses were observed at the 300 mg/kg dose of EAA, which implies that although consumption of the leaf extract may not predispose users to hepatotoxicity, haematotoxicity, and hyperlipidemia, it should be taken with caution during pregnancy due to possible risk of embryotoxicity at concentrations higher than the therapeutic dose. Copyright

Euphorbia tirucalli aqueous extract induces cytotoxicity, genotoxicity and changes in antioxidant gene expression in human leukocytes

Euphorbia tirucalli, popularly known as “avelos”, is a toxic plant used as tea in Brazilian folk medicine as an antibacterial, antiviral and anticarcinogenic agent. However, there is no scientific report about its potential toxicity in human cells. Therefore, the objective of the present study was to evaluate the in vitro genotoxicity and cytotoxicity of aqueous extracts of E. tirucalli in human leukocytes using a comet assay and trypan blue exclusion assay, respectively. In addition, the effect of E. tirucalli on the osmotic fragility was investigated in human erythrocytes. The expressions of selected antioxidant mRNA genes (SOD2, CAT and GPx4) as well as tumor protein p53 (TP53) were evaluated by qRT-PCR. Exposure of human leukocytes to high concentrations of aqueous extracts of E. tirucalli (100–150 μg mL−1) caused a significant increase in DNA damage. Leukocyte viability was decreased in the presence of 50–150 μg mL−1E. tirucalli extract. E. tirucalli did not change the osmotic fragility of human erythrocytes. High concentrations of E. tirucalli (10–50 μg mL−1) significantly up-regulated the mRNA of SOD2 and CAT and decreased the mRNA of GPx4 expression in human leukocytes. In addition, the mRNA gene expression of SOD2 was down-regulated at the highest concentration tested (150 μg mL−1). In summary, based on the results of genotoxicity observed in our study, we recommend caution regarding the acute or chronic use of this homemade preparation. Taken together, our results suggest that the aqueous extract of E. tirucalli induces genotoxicity and cytotoxicity in human leukocytes, possibly by interacting with the antioxidant enzyme system, thereby, increasing the formation of ROS and decreasing the cellular tolerance level to chemical constituents of this plant.

Impairment of Hepatic and Renal Functions by 2,5-Hexanedione Is Accompanied by Oxidative Stress in Rats

2,5-Hexanedione (2,5-HD) is the toxic metabolite of n-hexane which is widely used as solvent in numerous industries. The present study elucidated the precise mechanism of 2,5-HD in hepatorenal toxicity by determining the involvement of oxidative stress in rats. Adult male Wistar rats were exposed to 0, 0.25, 0.5, and 1% 2,5-HD in drinking water for 21 days. Exposure to 2,5-HD caused liver and kidney atrophy evidenced by significant elevation in serum aminotransferases, alkaline phosphatase, albumin, bilirubin, urea, creatinine, and electrolytes levels compared with control. The marked dose-dependent increase in total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL) was accompanied with significant decrease in high-density lipoprotein (HDL) levels in 2,5-HD-exposed animals when compared with the control. Administration of 2,5-HD significantly diminished glutathione (GSH) level but increased the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione-S-transferase (GST) concomitantly with marked elevation in hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels in liver and kidney of the treated groups compared with control. These findings suggest that undue exposure to 2,5-HD at environmentally relevant levels may impair liver and kidney functions through induction of oxidative stress.

Redox status of the testes and sperm of rats following exposure to 2,5-hexanedione

Objectives: Exposure to 2,5-hexanedione (2,5-HD) is well known to be associated with reproductive dysfunctions in both humans and animals. However, the role of oxidative stress in 2,5-HD-induced toxicity in testes and sperm has not yet been studied. Methodology: The present study investigated the influence of 2,5-HD on antioxidant systems in the testes and epididymal sperm of rats following exposure to 0, 0.25, 0.5, and 1% 2,5-HD in drinking water for 21 consecutive days. Results: Administration of 0.5% 2,5-HD significantly (P < 0.05) decreased epididymis weight, whereas 1% 2,5-HD-treated rats showed significantly decreased body weight, testis, and epididymis weights compared with the control group. Exposure to 2,5-HD caused a significant dose-dependent increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase in both testes and sperm compared with the control group. Moreover, 2,5-HD-exposed rats showed significant decrease in glutathione-S-transferase activity and glutathione level with concomitant significant elevation in the levels of hydrogen peroxide and malondialdehyde in both testes and sperm. Testicular and epididymal atrophy with significant, dose-dependent, decrease in epididymal sperm number, sperm motility, and viability were observed in 2,5-HD-treated rats. Conclusion: 2,5-HD exposure impaired testicular function and sperm characteristics by disruption of the antioxidant systems and consequently, increased oxidative stress in the treated rats.

Sperm characteristics, antioxidant status and hormonal profile in rats treated with artemisinin.

The indiscriminate use, abuse and patients’ noncompliance to normal prescription of artemisinin and its derivatives are a common practice during the treatment for drug‐resistant malaria parasites in most developing countries. This study investigated the influence of artemisinin on the testicular and epididymal sperm antioxidant systems as well as on the plasma levels of hormones from the pituitary and thyroid components of the brain–pituitary–testicular axis. Oral exposure of rats to 0, 7 and 35 mg kg−1 artemisinin for 7 days showed that the testicular antioxidant status at both therapeutic dose (7 mg kg−1) and overdose (35 mg kg−1), and the sperm antioxidant status at therapeutic dose of artemisinin remained unaffected compared with control. However, increased hydrogen peroxide and lipid peroxidation levels were accompanied by a concomitant decrease in glutathione peroxidase and glutathione‐S‐transferase activities as well as glutathione level in spermatozoon of rats administered with overdose of artemisinin. While plasma levels of all the hormones investigated remained unaffected, severe epididymal degeneration with concomitant decrease in sperm quantity and quality was observed in rats treated with overdose of artemisinin compared with control. Overall, induction of oxidative stress in the epididymis, but not in the testes, could cause reproductive deficits in individuals unduly undergoing artemisinin therapy.

Biochemical and haematological evaluation of repeated dose exposure of male Wistar rats to an ethanolic extract of Artemisia annua.

Artemisia annua is widely used for the treatment of malaria and other disorders. In a previous study, the artemisinin concentration in the dry leaves of A. annua grown under humid tropical conditions was determined to be 1.098% using reversed phase high performance liquid chromatography. In the current study, biochemical and haematological evaluations of ethanolic leaf extracts derived from such plants (EAA) were carried out in 20 male Wistar rats. Rats were divided into four study groups of saline‐treated (control) and test groups exposed orally to graded doses of EAA for 28 days. The results showed that the liver function and haematological indices, and testosterone levels were not adversely affected. High density lipoprotein ‐cholesterol was reduced at 100 mg/kg of EAA, atherogenic index as well as low density lipoprotein ‐cholesterol was raised, and glucose concentration was reduced significantly at the 100 and 200 mg/kg of EAA (p < 0.05). In addition to serving as a possible antidiabetic agent, EAA may not predispose users to hepatotoxicity, haematotoxicity and testicular toxicity. However, due to the possible risk of atherosclerosis, we advise that the plant extract should be taken with caution in people with atherosclerotic condition. Copyright

Suppression of the brain-pituitary-testicular axis function following acute arsenic and manganese co-exposure and withdrawal in rats

Despite the fact that most environmental exposures to metals do not occur in isolation, the combined effects of metal mixtures on brain-pituitary-gonadal axis are poorly known. The present study investigated the impacts of co-exposure to arsenic (As) and manganese (Mn) on sperm characteristics, reproductive hormones and selected oxidative stress indices in the brain, testes and epididymis of rats following exposure for 15 consecutive days to 60mg/L of AsO2Na and 30mg/L of MnCl2 in drinking water. The results showed that while the brain weight remained unaffected, the fluid intake and the weights of testes and epididymis significantly (p<0.05) decreased in all the treatment groups. A significant decrease in the body weight gain when compared with control was noted only in the co-exposed rats. Moreover, the significant decreases in the antioxidant status in brain, testes and epididymis as well as in the circulatory concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone were similar following separate or combined exposure of rats to As and Mn. The marked oxidative damage in the investigated tissues was accompanied by a significant decrease in the sperm quantity and quality in all the treated rats when compared with the control. Interestingly, most of the parameters determined immediately after the exposure period persisted in rats from the withdrawal experiment. Collectively, co-exposure to As and Mn suppressed the brain-pituitary-testicular axis function and the post-testicular events such as sperm function possibly via a mechanism involving persistent oxidative stress and endocrine disruption in the exposed rats.

Dietary consumption of monosodium L-glutamate induces adaptive response and reduction in the life span of Drosophila melanogaster

Adaptive response is the ability of an organism to better counterattack stress‐induced damage in response to a number of different cytotoxic agents. Monosodium L‐glutamate (MSG), the sodium salt of amino acid glutamate, is commonly used as a food additive. We investigated the effects of MSG on the life span and antioxidant response in Drosophila melanogaster (D. melanogaster). Both genders (1 to 3 days old) of flies were fed with diet containing MSG (0.1, 0.5, and 2.5‐g/kg diet) for 5 days to assess selected antioxidant and oxidative stress markers, while flies for longevity were fed for lifetime. Thereafter, the longevity assay, hydrogen peroxide (H2O2), and reactive oxygen and nitrogen species levels were determined. Also, catalase, glutathione S‐transferase and acetylcholinesterase activities, and total thiol content were evaluated in the flies. We found that MSG reduced the life span of the flies by up to 23% after continuous exposure. Also, MSG increased reactive oxygen and nitrogen species and H2O2 generations and total thiol content as well as the activities of catalase and glutathione S‐transferase in D. melanogaster (P < .05). In conclusion, consumption of MSG for 5 days by D. melanogaster induced adaptive response, but long‐term exposure reduced life span of flies. This study may therefore have public health significance in humans, and thus, moderate consumption of MSG is advocated by the authors.