D-Penicillamine prolongs survival and lessens copper-induced toxicity in Drosophila melanogaster

ABSTRACT

D-penicillamine (DPA) is an amino-thiol that has been established as a copper chelating agent for the treatment of Wilson’s disease. DPA reacts with metals to form complexes and/or chelates. Here, we investigated the survival rate extension capacity and modulatory role of DPA on Cu²⁺-induced toxicity in Drosophila melanogaster. Adult Wild type (Harwich strain) flies were exposed to Cu²⁺ (1 mM) and/or DPA (50 μM) in the diet for 7 days. Additionally, flies were exposed to acute Cu²⁺ (10 mM) for 24 h, followed by DPA (50 μM) treatment for 4 days. Thereafter, the antioxidant status [total thiol (T-SH) and glutathione (GSH) levels and glutathione S-transferase and catalase activities] as well as hydrogen peroxide (H₂O₂) level and acetylcholinesterase activity were evaluated. The results showed that DPA treatment prolongs the survival rate of D. melanogaster by protecting against Cu²⁺-induced lethality. Further, DPA restored Cu²⁺-induced depletion of T-SH level compared to the control (P < 0.05). DPA also protected against Cu²⁺ (1 mM)-induced inhibition of catalase activity. In addition, DPA ameliorated Cu²⁺-induced elevation of acetylcholinesterase activity in the flies. The study may therefore have health implications in neurodegenerative diseases involving oxidative stress such as Alzheimer’s disease.

Introduction

Copper is a transition metal strategic to cellular metabolism especially in the brain.¹ Copper tissue levels in the human brain and in the liver are about 5 μg/g, while 0.3–0.5 μM copper concentration is found in the cerebrospinal fluid.^2,3 Copper exists in two oxidation states making it an essential component of key enzymes involved in various biological processes including oxidative phosphorylation, modulation of neurotransmission etc.^4,5 In such enzymes, copper is found as prosthetic groups or bound to enhance activity as seen in superoxide dismutase (Cu-Zn-SOD), dopamine–β-hydroxylase, ceruloplasmin and cytochrome oxidase.^6,7 Importantly, free copper concentrations is tightly regulated in healthy cells especially at very low levels⁴; however, copper can be deleterious at high concentrations seemingly due to its ability to transit between oxidation states (Cu⁺ to Cu²⁺).^8–10 Sources of unrestrained intake of copper include foods such as liver, seafood, nuts, whole grains and dried fruits. Residues of pesticides in contaminated soils and drinking water as well as leaching of copper from corroded water pipes also increase the immoderate consumption of copper.^11–13

Copper is transported via a coordinated assembly of proteins such as copper transporter 1 (CTr1) and nonspecific divalent metal transporter 1 (DMT1).¹⁴ The efflux of copper is in turn mediated by copper ATPases, such as copper transport protein alpha (ATP7A) and beta (ATP7B).¹⁵ Other mediators of copper transport include copper chaperones such as Atx1p/Atox1 which enhance the delivery of copper to ATPpase copper requiring proteins, and Cox 17 which transports copper to the mitochondrial enzyme cytochrome c oxidase.^16–18

Copper dysmetabolism has been associated with toxic effects believed to be caused by oxidative stress and has been implicated in the pathogenesis of neurodegenerative disorders such as Wilson’s disease (WD), Menkes disease, Alzheimer’s disease (AD), Parkinson’s disease and amyotrophic lateral sclerosis.¹⁹

Oxidative stress and damage are implicated in the pathogenesis and progression of AD²⁰; however, several controversies and speculations have been made on the precise role of Aβ peptide and copper ions.^21,22 In some spheres, adductions that Aβ toxicity is due to reactive oxygen species (ROS) generation in the presence of the Aβ-Cu (II) complex were made, while others debated an antioxidant role for Aβ.^23,24 Given the neurotoxic potential of copper and the associated interactions, strategies that disrupt the Cu–Ab interaction by using chelators was proposed as a therapeutic strategy, which is an emerging trend in current research. Hence, there is immense need to develop such a suitable copper chelator that could prevent amyloid-β aggregation by effectively sequestering extra Cu²⁺ ions. Similar effects may suffice in other neurodegenerative diseases.

D-Penicillamine (DPA; β-β-dimethylcysteine or 3-mercapto-D-valine) is a sulfhydryl-containing amino acid and a degradation product of penicillin (Fig. 1). DPA is used mainly as a chelating agent in copper poisoning and in the treatment of WD.²⁵ DPA has the ability to function as either a mono-, bi- or tridentate ligand. The thiol of DPA binds to Cu (I) and eliminates it through urine both in normal people and in patients with WD.^26,27 Despite the fact that DPA has been reported to efficiently reduce excess Cu in the system,²⁸ other authors found that in the process of Cu chelation by DPA, Cu (II) is reduced to its Cu (I) form, which leads to the generation of hydrogen peroxide (H₂O₂) and other ROS.^28–30 We therefore envision that investigating the chelating property of DPA in different animal model such as invertebrates like Drosophila melanogaster may shed more light on the protective ability of DPA in copper-induced toxicity.

Figure 1.

Chemical structure of D-penicillamine (A) and D-penicillamine disulfide (B).

Drosophila melanogaster is a nonmammalian model that has been extensively employed as an in vivo model system to characterize the effects of environmental neurotoxic metals.^31–36 Also, the fly expresses proteins involved in the metabolism of biometals such as ferritin,³⁷ transferrin,³⁸ iron regulatory proteins,³⁸ DMT1³⁹ and DmATP7^40,41 similar to human. Consequently, we investigated the protective potential of DPA on copper-induced toxicity in D. melanogaster.

Materials and Methods

Chemicals

The 1-chloro-2, 4-dinitrobenzene, 5,5′-dithiobis (2-nitrobenzoic acid), acetylthiocholine iodide, were purchased from Sigma (USA). Copper sulfate and DPA were procured from A K Scientific, 30023 Ahern Ave, Union City, CA 94587, USA. Other reagents were commercial products of the highest analytical grade.

Drosophila melanogaster stock and culture

Drosophila melanogaster wild-type (Harwich strain) flies were obtained from the National Species Stock Center (Bowling Green, OH, USA). The flies were maintained and reared in Biochemistry Department, University of Ibadan, Nigeria on cornmeal medium containing 1% w/v brewer’s yeast, 2% w/v sucrose, 1% w/v powdered milk, 1% w/v agar and 0.08% v/w nipagin at constant temperature and humidity (22–24°C; 60–70% relative humidity) under 12 h dark/light cycle conditions.

Cu²⁺acute and chronic exposures and DPA treatment

Copper sulfate (CuSO₄) and DPA were added into the medium at a final concentration of Cu²⁺ 1 mM and/or DPA 50 μM, respectively. Wild type D. melanogaster of both genders of 1–3 days old were divided into four groups with 50 flies/vial: (1) Control; (2) DPA (50 μM); (3) Cu²⁺ (1 mM); (4) Cu²⁺(1 mM) plus DPA (50 μM) for 7 days. Further, flies were divided into three groups (n = 5) for acute Cu²⁺ study: (1) control; (2) Cu²⁺ (10 mM); (3) Cu²⁺(10 mM) plus DPA (50 μM). Here, flies were first exposed to Cu2+ (10 mM) for 24 hrs followed with DPA (50 μM) treatment for 4 days. The choice of 1 mM Cu²⁺ was informed by a study carried out by Halmenschelager and Rocha⁴², Abolaji et al.⁴³ and 50 μM DPA from Bonilla-Ramirez et al.⁴⁴ (Fig. 2).

Figure 2.

Treatment regimen and duration of Cu²⁺ and DPA treatments. (A): Flies were exposed to DPA and/or Cu2+ for 7 days for the determination of survival rate and biochemical assays. (B): Flies were first exposed to Cu2+ for 24 hr followed with DPA treamtment for 4 days.

In vivo assays

Survival rates determination

The survival rate was determined daily by counting the number of living flies until the end of each experimental period (7 days). A total of 200 flies per group were included in the survival data (40 flies/vial, n = 5) as previously reported.⁴⁵

Ex vivo assays

Preparation of samples for biochemical assays

For the biochemical assays, flies were anaesthetized once, weighed, homogenized in 0.1 M potassium phosphate buffer, pH 7.4 (1: 10 (flies/volume (μL)), and centrifuged at 10 000 g for 10 min at 4°C. The supernatant was separated from the pellets into Eppendorf tubes, kept at −20°C and used to determine acetylcholinesterase (AChE), catalase and glutathione S-transferase (GST) activities as well as T-SH and H₂O₂ levels. Notably, all the assays were carried out in duplicates for each of the five replicates of copper sulfate and the DPA concentrations.

Determination of selected oxidative stress indices and AChE activity

We determined protein concentration using the method described by Lowry et al.⁴⁶ Total thiol content was carried out with the method of Ellman.⁴⁷ GST activity was determined according to the method of Habig and Jakoby.⁴⁸The activity of catalase was determined using the method of Aebi.⁴⁹ We evaluated AChE activity using the method of Ellman et al.⁵⁰ Hydrogen peroxide level was determined according to the method of Wolff.⁵¹

Statistical analysis

The Kaplan–Meier’s method was used to analyze the survival rate and comparisons were made with the log-rank tests. The data are presented as the mean ± SEM. A One-way Analysis of variance was used to assess the significant differences among multiple groups followed by Dunett’s post hoc test. Statistically significant differences indicated at P < 0.05, using the GraphPad Prism 5.0 software.

Results

Survival rates of flies exposed to Cu²⁺ and DPA

Cu²⁺ (1 mM) exposure resulted in a significant dose-dependent decrease in survival of flies compared with the control groups (P < 0.05). The Cu²⁺-induced mortality was ameliorated significantly by DPA (Fig. 3), indicating DPA’s ability to prolong survival and protect against Cu²⁺-induced mortality.

Figure 3.

Effect of DPA and Cu²⁺ on survival of Drosophila melanogaster. This was to investigate the ability of DPA to prolong survival. The p values (log-rank tests) for each group include: Ctrl versus DPA 50 μM (P = 0.1214), Cu²⁺ 1 mM (P < 0.05), DPA50 μM + Cu²⁺ 1 mM (P < 0.05). The maximum lifespan in each group represents the percentage of surviving flies.

DPA modulates chronic Cu²⁺-induced accumulation of H₂O₂ level, depletion of T-SH and inhibition of catalase activity in D. melanogaster

The exposure of D. melanogaster to Cu²⁺ (1 mM) and DPA for 7 days indicated that DPA had no effect on Cu²⁺-induced inhibition of GST activity (Fig. 4C, P < 0.05) in the flies. However, DPA restored Cu²⁺-induced depletion of total thiol level (Fig. 4A) and ameliorated Cu²⁺-based inhibition of catalase activity (Fig. 4E) in D. melanogaster (P < 0.05).

Figure 4.

Effects of DPA and chronic Cu²⁺ exposure on oxidative stress parameters. Total thiol level (A), glutathione level (B), glutathione S-transferase activity (C), hydrogen peroxide level (D), catalase activity (E) in Drosophila melanogaster. Values are expressed as mean ± standard error of mean (n = 5). Significant differences from the control are indicated by asterisk (P < 0.05). Significant differences from the Cu²⁺-treated group are indicated by hash (P < 0.05).

DPA effect on AChE activity in D. melanogaster

We noted that, Cu²⁺ (1 mM) caused a marked elevation in AChE activity in flies, while flies co-treated with 50 μM DPA had AChE activity restored to level comparable to DPA-treated group (P < 0.05; Fig. 5).

Figure 5.

Effect of DPA and chronic Cu²⁺ exposure on acetylcholinesterase activity. Effect of curcumin on Cu²⁺-induced toxicity in treated flies. AChE activity in flies after treatment for 7 days. Values are expressed as mean ± standard error of mean. Significant differences from the control are indicated by asterisk (P < 0.05). Significant differences from the Cu²⁺-treated group are indicated by hash (P < 0.05).

DPA moderates acute Cu²⁺-induced depletion of T-SH in D. melanogaster

We observed significant depletion of total thiol level (Fig. 6A, P < 0.05) in Cu²⁺-exposed flies along with significant inhibition of catalase activity (Fig. 6E, P < 0.05) when compared with the control group. However, cotreatment with DPA only augmented total thiol level without ameliorating Cu²⁺ (10 mM)-induced inhibition of catalase and GST activities as well as reduction of GSH (Fig. 6B) and elevation of H₂O₂ (Fig. 6D) levels and increased catalase activity (Fig. 6E) in the flies.

Figure 6.

Effects of DPA and Cu²⁺ on oxidative stress and antioxidant parameters. Total thiol level (A), glutathione level (B), activity (C), hydrogen peroxide level (D) and catalase activity (E) in Drosophila melanogaster. Values are expressed as mean ± standard error of mean (n = 5). Significant differences from the control are indicated by asterisk (P < 0.05). Significant differences from the Cu²⁺-treated group are indicated by hash (P < 0.05).

Discussion

In this study, we demonstrated that exposure of D. melanogaster to Cu²⁺ increased mortality and induced oxidative stress. Comparable to xenobiotic-induced neurotoxicity as observed in rotenone,⁵²paraquat⁵³ and MPTP⁵⁴ in Drosophila, metals can promote neuronal loss of the dopaminergic neurons. Specifically, elevated copper levels resulted in the generation of ROS, eliciting oxidative damage.⁵⁵ The molecular mechanisms responsible for the uptake, efflux, distribution and regulation of metals are conserved in D. melanogaster.^55–58 Thus, this study was carried out to understand if DPA, a notable copper chelator, could protect D. melanogaster against Cu²⁺-induced toxicity.

Although, previous studies have highlighted the role of DPA in reducing serum oxidative stress in AD patients,⁵⁹ improving antioxidant capacity in WD⁶⁰ and depleting body copper load,⁶¹ these studies have not addressed the possible implications of copper toxicity or likely exposure of patients to copper. Our study addressed copper-induced toxicity in an invertebrate model whose metabolic networks are capable of genetic manipulation.

Flies exposed to Cu²⁺ showed decreased survival rate during the 7-day exposure period, while cotreatment of Cu²⁺ with DPA ameliorated Cu²⁺-induced lethality. This result indicated a possible antioxidant potential of DPA that possibly decreased the observed premature mortality of D. melanogaster exposed to Cu²⁺.⁶²

We observed that DPA did not ameliorate Cu²⁺-induced accumulation of H₂O₂ and inhibition of catalase activity in the acute study in D. melanogaster. This observation may be due to the reported interaction between DPA and copper resulting in the formation of multivalent DPA-copper complex (Cu₈^ICu₆^II (DPA)¹²Cl)⁵⁻,⁶³ which partly explains the cause for the overproduction of H₂O₂.⁶³ Catalase is a heme-containing enzyme that catalyzes the dismutation of H₂O₂ to molecular oxygen and water in order to minimize oxidative stress-mediated toxicity.⁶⁴ One limitation of the present study was that we did not carry out the level of copper in the flies. Nevertheless, the H₂O₂ accumulation from the direct effect of copper redox state transitions was not detoxified by catalase, due to Cu²⁺(10 mM)-induced inhibition of its activity in the acute study.

Furthermore, DPA did not ameliorate Cu²⁺-induced inhibition of GST activity. GSTs are enzymes with cysteine-rich domains^64,65 involved in the elimination of reactive molecules via conjugation with GSH. Thus, the observation that Cu²⁺ inhibited GST activity is as a result of copper-mediated oxidation of its thiol groups, thus modifying its native structure and conformation resulting in reduced activity. Further evidence indicated that copper interacts with GSH and forms Cu^(I)-[GSH]⁽²⁾ complex, which reduces molecular oxygen into superoxide.⁶⁵ However, DPA forestalled Cu²⁺-induced depletion of T-SH presumably due to the buffering capacity of other thiol-containing proteins in the system.

Moreover, DPA attenuated Cu²⁺ (1 mM)-induced elevation of AChE activity. AChE is an enzyme that participates in cholinergic neurotransmission by hydrolyzing acetylcholine to acetate and choline. It plays important role in learning, memory and locomotor activities. Cognitive deficits and behavioral functions can be easily modulated by cholinergic signaling and this has been shown to be altered by Cu²⁺.^66–68 Thus, DPA may modulate locomotor activity and cognitive deficits in D. melanogaster.

Conclusion

In summary, we report that Cu²⁺ exposure significantly abrogated survival and increased oxidative stress in D. melanogaster. We also showed that Cu²⁺ impacted the cellular antioxidant status negatively by generation of oxidative stress. However, DPA, via its chelating potential, ameliorated Cu²⁺ (1 mM)-induced toxicity, but partially lessened Cu²⁺ (10 mM)-induced toxicity in D. melanogaster. Further studies that aim to evaluate the expression of notable copper transporters ATP7A and CTR1 so as to estimate transportation activity of copper in D. melanogaster as well as studies quantifying the magnitude and effects of the copper-DPA complex are called for. Also, studies that aim at assessing the effect of DPA in Drosophila transgenic models of WD are also warranted. This study may thus be of public health significance as it provided further insights on DPA’s protective mechanisms against copper-induced toxicity.

Conflict of Interest

The authors declare that they have no conflict of interest.