Introduction
Chlorogenic acid (CA) is a polyphenol from the family of hydroxycinnamic acid. It is one of the main polyphenols in the human diet with many health-promoting properties. CA can be found in foods and herbs such as coffee beans, apples, tea, grapes, tomato etc. 12. Furthermore, it has been found that chlorogenic acid has antioxidant, anti-inflammatory, anticancer, antilipidemic, antidiabetic, antihypertensive and anti-neurodegenerative activities 345678.
Rotenone (ROT) is a commonly used natural pesticide from the roots of Derris elliptica and Lonchocarpus utilis9. It can cross the blood brain barrier, due to its lipophilic property and causes neurotoxicity 1011. ROT blocks the mitochondrial electron transport chain through the inhibition of complex I which causes cytotoxicity due to altered redox cycling and oxidative stress 12. It has been observed that high doses of ROT can induce generalized neurodegeneration, therefore, investigations have been carried out on low-dose systemic administration of this compound in rodents. In this condition, there is slow and specific loss of dopaminergic neurons and impaired mobility in animals treated with ROT, which are attributes of Parkinson’s disease 13.
Physical aberrations characterized by Parkinson’s disease (PD) (a type of neurodegenerative disease) include progressive impairment in mobility ability such as bradykinesia, tremor and rigidity. Studies have shown that PD resulted from the loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) and the formation of intra neuronal proteinaceous inclusions called Lewy bodies (LBs) in affected brain areas 14151617. Moreover, the pathology involves protein aggregates apart from LBs, various neurotransmitters and extensive regions of the nervous system 18.
In this study, using ROT-induced toxicity in Drosophila melanogaster as a model for neurodegeneration, the ameliorative effect of CA was evaluated.
Materials and Methods
Chemicals
All chemicals were of analytical grade. ROT and CA were procured from AK Scientific, 30023 Ahern Ave, Union City, CA 94587, United State of America.
Drosophila Melanogaster Culture
Drosophila melanogaster (Harwich strain) obtained from the National Species Stock Centre, Bowling Green, Oklahoma, U.S.A., were maintained and reared in the Drosophila Laboratory, Department of Biochemistry, University of Ibadan, Oyo State, Nigeria on cornmeal medium containing brewer's yeast (1% w/v), agar-agar (1% w/v), and nipagin (preservative, 0.08% v/w) at constant temperature (23 ± 2 °C) under 12 h dark/light cycle.
Rotenone and Chlorogenic Acid Exposure and Survival Rate Analyses
In order to determine the appropriate concentrations and duration of exposure to ROT and CA to be used for the main experiment, D. melanogaster (both genders, 1 to 3 days old) were allocated into three (3) groups of five vials with 50 flies each and administered ROT (0- control( 2% ethanol), 250, and 500 µM doses respectively) for 28 days for survival assay while CA was exposed to another six (6) groups of five vials with 50 flies each (0- control( 2% ethanol), 7.5, 15, 30, 45, and 60 mg/kg diet respectively) for 28-day longevity assay. For both assays, daily mortality was recorded, and data were analyzed and plotted as percentage of live flies. The effect on antioxidant status of exposure of six (6) groups of five vials with 50 flies each to CA (0- control( 2% ethanol), 7.5, 15, 30, 45, and 60 mg/kg diet respectively) was determined by assaying in the homogenate for catalase (CAT), glutathione-S-transferase (GST), hydrogen peroxide (H2O2), total thiol and reduced glutathione (GSH) after 7 days. Based on these data, 30 mg/kg diet of CA as well as 500 µM dose of ROT were selected for the study and used to determine the effect on biochemical parameters; acetylcholinesterase (AChE) activity (a marker for neurotoxicity) and oxidative stress-antioxidant status ((H2O2, total thiol, GSH levels, and CAT and GST activities), as well as the rate of emergence of offspring and negative geotaxis.
Determination of Negative Geotaxis and Rate of Emergence of Offspring
Locomotor performance of CA and ROT-treated flies were investigated using the negative geotaxis assay 1920. Emergence rate of D. melanogaster offspring after exposure and co-exposure to CA and ROT were evaluated as previously described by Farombi et al. 21.
Preparation of Sample for Biochemical Assays
For the determination of biochemical assays, 50 flies (of both gender)/ vial for 5 vials each per group, were exposed as described in each of the following groups: Group A (control)-ethanol (2%), Group B- CA only (30 mg/kg diet), Group C- ROT only (500 µM) and Group D- 500 µM ROT + 30 mg/kg diet CA for 7 days. At the end of the treatment period, flies were anaesthetized using CO2, weighed, and homogenized in 0.1 M phosphate buffer, pH 7.0 (ratio of 1 mg:10 ml), and centrifuged at 4000 g for 10 min at 4oC in a Thermo Scientific Sorval Micro 17R centrifuge. Subsequently, the supernatants were separated from the pellets into labeled Eppendorf tubes and used for the determination of the activities of AChE, GST, CAT, and total thiol, H2O2 and GSH content. All the assays were carried out in duplicates for each of the five replicates.
Determination of Biochemical Parameters
Estimation of Protein Concentration
The concentration of protein was evaluated by the method of Lowry et al.22.The sample (25 µl, 1:10 dilution) was mixed with 135 µl of distilled water, and added to 400 µl of alkaline copper sulphate reagent (Lowry reagent) followed by 15 minutes incubation time. Folin ciocalteau solution (1:5 dilution) was added and they were then incubated at room temperature for 20 mins. The absorbance was measured at a wavelength of 660 nm against blank and protein values were estimated from the standard curve of bovine serum albumin (BSA).
Estimation of Total Thiol Level
The method of Ellman 23 was used to determine total thiol content. The reaction mixture consists of 510 μl of 0.1 M phosphate buffer (pH 7.4), 20 μl of sample, 35 μl of 1 mM 5′,5′ – dithiobis- 2- nitrobenzoate (DTNB) and 35 μl of distilled water. The mixture was incubated for 30 mins at room temperature and the absorbance was measured at 412 nm.
Estimation of Hydrogen Peroxide Level
The level of H2O2 in the treatment was determined using the method of Wolff 24. The reaction mixture consist of FOX 1 (10 ml of 100 mM xylenol orange, 50 ml of 250 mM ammonium ferrous sulfate, 10 ml of 100 mM sorbitol, 5 ml of 25 mM H2SO4 and 30 ml of distilled water) mixed with the sample. Incubation was carried out for 30 mins at room temperature and the absorbance was measured at 560 nm. The values, which were deduced from the standard curve, were expressed in micromole per milligram protein.
Estimation of Reduced Glutathione Level
The estimation of the level of GSH was carried out with the method of Jollow et al.25 with little modifications. Sulphosalicyclic acid (4%) in the ratio of 1:1 was used to precipitate the supernatant. The samples were incubated for 1 h at 4oC and then centrifuged at 5000 rpm for 10 mins at 4oC. The reaction mixture consists of 550 µl of 0.1 M phosphate buffer, 100 µl of supernatant and 100 µl of DTNB. The absorbance was read at 412 nm and the results were expressed as moles of GSH/gram tissue.
Estimation of Glutathione-S-Transferase Activity
The method of Habig and Jakoby 26 was used to assess the activity of GST. The reaction mixture comprised of 270 μl of a solution consisting of 20 μl of 0.25 M potassium phosphate buffer, pH 7.0, with 2.5 mM ethylene diamine tetra acetic acid (EDTA), 10.5 ml of distilled water and 500 μl of 0.1 M GSH at 25 °C),10 μl of 25 mM 1- chloro-2,4- dinitrobenzene (CDNB; as substrate) and 20 μl of sample (1:5 dilution). The mixture was monitored for 5 min with 10 s intervals at 340 nm using a spectrophotometer.
Determination of Catalase Activity
CAT activity was assessed with the method of Aebi 27. The reaction mixture consists of 50 mM of phosphate buffer (pH 7.0), 300 mM H2O2 and sample (1:50 dilution). The loss in absorbance of H2O2 was monitored for 2 min at 240 nm. The result was used to calculate CAT activity and expressed as μmol of H2O2 consumed per minutes per milligram of protein.
Determination of Acetyl Cholinesterase Activity
AChE activity was evaluated using the method of Ellman et al.28. The reaction mixture contained 135 μl of distilled water, 20 μl of 100 mM potassium phosphate buffer (pH 7.4), 20 μl of 10 mM DTNB, 5 μl of sample, and 20 μl of acetylthiocholine (8 mM). The reaction was monitored at a wavelength of 412 nm (for 5 min, 15 s intervals) in a SpectraMax microplate reader (Molecular Devices, USA). The data were expressed thereafter in μmol/min/mg protein.
Statistical Analysis
All of the experiments were replicated at least two times. The data are presented as the mean ± standard error of mean (SEM). To assess the significant differences among multiple groups under various treatments, One-way analysis of variance (ANOVA) was used. For all of the assays, differences with p-value < 0.05 were considered statistically significant.
Discussion
In this study, it was demonstrated in Drosophila melanogaster that CA ameliorated the deleterious effects of ROT on AChE and GST activities, total thiol and GSH levels, and climbing and eclosion rates.
PD is an age-related neurodegenerative disease affecting more than 1% of the human population. It is the second most common neurodegenerative disorder with prevalence increasing with age, mostly among population above 60 years 29. The symptoms of PD include progressive impairment in locomotive ability, tremor, rigidity, and bradykinesia 17. Currently, a combination of genetic changes and environmental factors has been observed to be responsible for this condition, though controversially 3031. Some of the environmental factors is the exposure to chemicals in pesticides and herbicides such as ROT, permethrin, MPTP (1-methyl 4-phenyl 1, 2, 3, 6-tetrahydropyridine), organochlorines, paraquat and 2, 4-dichlorophenoxyacetic acid 323334. In this study, ROT was used to induce Parkinsonism in Harwich strain of Drosophila melanogaster.
Currently, the modulatory role of several nutraceuticals to attenuate endogenous oxidative stress status has been considered as an effective approach to achieve neuroprotection 353637. As stated earlier, CA is a phenolic compound found in food of plant origin which has anti-diabetic, antioxidant, anti-inflammatory, anticancer, antilipidemic, and anti-neurodegenerative properties 345678. Moreover, studies have shown that consumption of diets rich in antioxidant property contribute to extension of lifespan in an organism 38. The fly-survival and longevity experiment based on CA showed that among the concentration range tested (7.5- 60 mg/kg diet), 30 mg/kg of CA was the most effective (Figure 1). Therefore, in regard to the ingestion of CA, moderate levels would be more beneficial to increase longevity. Moreover, CA significantly reduced the level of H2O2 with the 30 mg/kg diet and did not show any adverse effect on the activities of CAT and GST as well as the levels of total thiol and GSH (Figure 2).
In this study, it was observed that 28-day exposure of D. melanogaster to ROT elicit alterations in the levels of antioxidant enzymes activities (such as CAT and GST), perturbations in reduced total thiol and GSH levels, increased H2O2 level, induced behavioural deficit as well as decreased AChE activity. All the aforementioned detrimental effects observed (with the exception of H2O2 level) were mitigated by CA (Figures 4 and 5).
CAT catalyzes the conversion of H2O2 to water (H2O) and oxygen. The most common one is a monofunctional heme-containing enzyme 39. It is an intracellular antioxidant enzyme that protects biological macromolecules from oxidation by H2O239. This study shows an elevated CAT activity in flies exposed to ROT which was mitigated to levels comparable to control by CA (Figure 4B). This reveals the modulatory role of CA in obtaining an optimum level of H2O2 molecule in the cell, which is also essential in cellular signaling and this is supported by the observation with the CA diets (Figure 2C).
H2O2 is a reactive oxygen species (ROS). Elevated levels of H2O2 indicate that a system is plagued by oxidative stress 39. In this study it was observed that there was reduction in the level of H2O2 in flies treated with CA alone. However, ROT caused a significant accumulation of H2O2 which was mitigated, but not significantly, by CA (Figure 4A). Antioxidants play protective roles by scavenging reactive oxygen species and reducing the insults of oxidative damage 40. Although CA did not significantly reduce H2O2 level in the co-administered flies, the reduction in CAT activity upon co-administration shows that CA has an ameliorative effect on ROT-induced toxicity but was not distinct enough on H2O2 level upon conclusion of the experiment.
Total thiols (protein and non-protein thiols) defend the system against oxidative damage 41. GSH functions as part of the first line of antioxidant defense system in living organisms. In conjunction with GSH-dependent enzymes, it helps to block the propagation of free radical chain reactions and detoxify deleterious ROS 42. GSH is an important antioxidant in living organisms that prevents oxidative damage to cellular components 424344. Depletion of cytosolic GSH in neurons is one of the hallmarks in PD 45. The decrease in levels of total thiols and GSH in the ROT challenged group evidenced from our study suggests a compromised defense system in D. melanogaster. However, this study shows that CA modulates ROT-induced reduction of thiol containing compounds, thus confirming its anti-oxidative capacity. Hence, CA may be an ameliorative agent against diseases associated with altered redox state of a system.
GST is a major phase II detoxification enzyme, which comprises of a large and multi-functional enzyme family primarily, involved in the detoxification of endogenous substrate by catalyzing the conjugation of the nucleophilic sulphur atom of GSH with a variety of reactive electrophilic exogenous compounds 344647. The result from our study shows that CA protected against ROT-induced inhibition of GST activity. Since GST catalyses the conjugation of GSH with electrophilic molecules, GSH was rapidly used up in order to protect the flies. Therefore, the ameliorative mechanism of CA in ROT-induced toxicity may be by further enhancing activity of thiol-containing proteins like GST.
AChE is a vital enzyme of the cholinergic system that modulates learning, sleep and wakefulness, memory, attention, stress response and locomotor activities. It hydrolyzes acetylcholine to choline and acetate thereby terminating cholinergic neurotransmission between synapses 4849. Our study revealed that CA significantly alleviated ROT-induced inhibition of AChE activity when compared with control. This finding suggests that the modulatory action of CA on AChE activity may improve cognitive impairment evident in neuro-related diseases.
In the negative geotaxis assay, it was observed that ROT decreased the climbing rate of the flies but the effect was mitigated in groups co-treated with CA. This shows that CA has an ameliorative potency against bradykinesia, which is one of the effects of ROT toxicity and a hallmark in PD. This is similar to the study conducted by Farombi et al. 21, in which kolaviron was able to modulate the effect of ROT in flies.
Flies’ emergence was highly increased in the group treated with CA only but a barren effect was recorded in the group treated with ROT only as compared with the control group. Also, substantial flies’ emergence was observed in the co-administered group treated. This shows that CA may have a highly ameliorative effect against reproduction deficit common in PD patients. This is in line with the study conducted by Namula et al. 50 in which CA, in addition with caffeic acid, was able to improve the quality of frozen-thawed boar sperm 50.