Toxicological Evaluation of Disulfiram , Copper Gluconate and Disulfiram / Copper Gluconate Combination on Renal Function in Rodents

This research work investigated and compared the chronic renal toxicological profile of disulfiram, copper gluconate and disulfiram/copper gluconate combination, in a 90-day timeand dose-dependent study in rodents. 88 rats weighing an average of 280 g divided into eleven groups consisting of 8 rats each were used for this experiment. The control groups received normal saline as placebo and 99.5% dimethyl sulfoxide (DMSO) (solvent control). Three oral doses (low, medium and high) of disulfiram (18.65 mg/kg, 37.3 mg/kg and 74.6 mg/kg), copper gluconate (3.75 mg/kg, 7.5 mg/kg and 15 mg/kg) and both drugs in combination were administered daily with those of the combination given 12 hours apart. Blood samples were collected via cardiac puncture in heparinised bottles and centrifuged, and the serum was decanted on 30, 45, 60 and 90 days for analysis. Renal function parameters—electrolytes (Na+, K+), urea and creatinine were evaluated. Results showed significant (p < 0.05) doseand time-dependent increase in electrolyte level (Na+, K+), blood urea and creatinine respectively. The results are all pointers to the development of renal failure. It therefore appears that the DSF/CG combination is nephrotoxic and this effect is dose-dependent and synergistic.


Introduction
Cancer, also termed malignant tumour or neoplasm, is a group of diseases involving abnormal cell growth with a potential to invade or spread to other parts of the body.In Nigeria, breast cancer, cervical cancer and prostate cancer are most prevalent amongst women and men respectively [1].Added to this, is the burden cancer imposes on the Niger delta region of Nigeria where this study is sited.Researchers attest to the fact that individuals in this region are at increasing risk of developing cancers as a result of oil exploration activities [2].This underscores the interest of the researchers in new drug treatment for cancers that would be readily available to low-income economies and affordable.Disulfiram is an old drug hitherto used for alcoholism.The addition of copper gluconate makes the combination a potentially effective and cheap means of treating cancer and is therefore being repurposed for use in cancer chemotherapy.Repurposing non-cancer related drugs with possible anti-tumoral activities is a promising strategy for identifying prospective new anti-cancer drugs in a cost-efficient and timesaving way [3]- [5].Repurposing disulfiram has recently become of interest because of its pre-clinically described anti-cancer effects against various human cancers, which include breast, cervical, colorectal, lung, melanoma, prostate as well as myeloma and leukaemia [6] [7].Disulfiram, a member of the dithiocarbamate family, possesses metal-binding properties [8] and is thought to inhibit superoxide dismutase resulting in inhibition of the angiogenic potential [9].In the presence of Cu (II), disulfiram is converted to the two-electron oxidized form of diethyldithiocarbamate, which is the active form in inducing cell death [10].Chen et al. [11] have reported that disulfiram could bind to cellular copper or zinc to form a complex that has a proteasome-inhibitory effect, which might contribute to its apoptosis-inducing effect.Diethyldithiocarbamate is a main physiological metabolite found after gastrointestinal uptake of disulfiram [12]- [14].Disulfiram and diethyldithiocarbamate are convertible into each other via a copper containing intermediate complex.Cen et al. [10] also proposed that the complex of two diethyldithiocarbamate molecules formed by redox active Cu (II), could be mainly responsible for the proapoptotic response to disulfiram.In a study on ovarian cancer cell lines, Papaioannou et al. [15] reported that when cell lines were tested using disulfiram alone and disulfiram with copper supplementation, disulfiram alone reduced cell survival of ovarian cancer cells at an optimum concentration even in the absence of copper supplementation, but supplementation with 1 µM copper chloride, increased the cytotoxic effect of disulfiram in all other ovarian cancer cells tested.
A major problem with most antineoplastic agents is the adverse effects that occur following their use.Renal failure in cancer patients is a common problem in oncology and this complication is frequently multifactorial in origin.Several antineoplastic agents are potentially nephrotoxic; previous renal impairment as well as combinations with other nephrotoxic drugs may increase the risk of nephrotoxicity during administration of chemotherapy [16].Also, exposure to heavy metals is potentially harmful.Because of its ability to reabsorb and accumulate divalent metals, the kidney is the first target organ of heavy metal toxicity.The extent of renal damage by heavy metals depends on the nature, the dose, the route and the duration of exposure [17].We set out to study the effect of disulfiram, copper gluconate and disulfiram/copper gluconate combination on the kidneys in rodents.

Methodology
88 male albino Swiss rats weighing an average of 280 g obtained from the Department of Pharmacology animal house were used for this study.The rats were bred and maintained under suitable conditions, allowed an acclimatization period of two (2) weeks, housed in hygienic cages in groups of four and allowed free access to feed obtained from vital feeds UAC PLC and water ad libitum.The beddings were changed and cages cleaned out on alternate days.Animals were handled according to Helsinki declaration on animal care.The animals were divided into 11 groups, each consisting of 8 rats each.The groups included those for treatment and the control groups.Drugs were administered orally via a 1ml syringe.

Chronic Toxicity Tests
This study spanned 3 months and was domiciled in the Department of Pharmacology, University of Port Harcourt, Animal House and Laboratory.A dose and time dependent toxicological evaluation of the effects of these individual drugs and their combinations on the renal profiles of rodents was evaluated.The rats were divided into eleven groups consisting of 8 rats each.Groups 1 and 2 served as control groups and the rats received normal saline as placebo and 99.5% DMSO (solvent control) respectively.Drugs were administered orally via a 1 ml syringe as 1/5 th , 1/10 th and 1/20 th of the LD 50 of disulfiram and copper gluconate at 373 mg/kg and 75 mg/kg respectively [18].

Collection of Samples
Two animals per group were sacrificed using diethyl ether anaesthesia and blood samples were obtained on days 30, 45, 60 and 90 for analysis via cardiac puncture.Blood samples were centrifuged at 3000 rpm for 15 minutes and serum separated from the cells.The samples were then assayed for Na + , K + , by flame photometry [20] and urea and creatinine using the Clinical Chemistry Autoanalyser RX Series by Randox Laboratories Limited, United Kingdom.

Stock Solutions
Stock solutions were prepared from 99.5% DMSO for disulfiram and distilled water for copper gluconate.Pure analytical grade samples, CAS No. 527-09-3 (98% min purity) and CAS No. 97-77-8 (98% min purity) obtained from Shijiazhuang Aopharm Import and Export Co. Limited China were used for the study.

Ethical Approval
Ethical approval was obtained from the University of Port Harcourt Research Ethics Committee.

Statistical Analysis
Statistical analysis was done using graph pad prism 5 statistical package and ANOVA for comparison of the means of the various groups.Results are expressed as means ± SEM.Test group results were compared with that of the control groups.A p-value < 0.05 was considered significant.

Results and Discussion
Anticancer agents are frequently associated with a variety of renal and electrolyte disorders.These drugs could affect the kidneys manifesting as an asymptomatic elevation of serum creatinine or acute renal failure.The kidneys are the major pathway for elimination of many antineoplastic agents as well as their metabolites.Therefore, renal impairment can result in delayed drug excretion and metabolism of anticancer agents resulting to increased systemic toxicity.Potassium, an essential intracellular, positively charged ion, is actively "pumped" in to the cell from surrounding extracellular fluid, while, sodium, is pumped out.This is necessary for proper fluid balance, and creates an electrical charge across the cell membrane.This is also the fundamental principle which allows nerves to conduct impulses and so communicate between cells and muscles to contract.Potassium is important to proper heart functioning.Hypokalaemia or hyperkalaemia quickly leads to electrolyte imbalance which affects all muscles, nerves and numerous key body functions.Increased potassium levels could be as a result of damage to the kidneys resulting in extrusion of the ions into the extracellular space [21].Results of the present study revealed that low dose disulfiram, copper gluconate and disulfiram/copper gluconate combination revealed significantly increased (p < 0.05) sodium ion levels (Table 1).Medium and high doses of all three therapeutic agents significantly increased (p < 0.05) sodium levels (Table 2 and Table 3).Low, medium and high doses of   all three therapeutic agents increased potassium levels significantly (p < 0.05) (Tables 4-6).This results point to an increased risk for the development of kidney failure perhaps due to direct toxic effects of the agents on the kidneys.
The blood urea nitrogen (BUN) test is a measure of the amount of nitrogen in the blood that comes from urea.It is used as a marker of renal function, though it is inferior to other markers such as creatinine because blood urea levels are influenced by other factors such as diet and dehydration [22].On urea levels, disulfiram at low dose showed no significant effect (p > 0.05) while copper gluconate and the disulfiram and copper gluconate combination caused a significant increase (p < 0.05) when compared to the control (Table 7).At the medium and high doses, our results revealed significant increase (p < 0.05) for disulfiram alone, copper gluconate alone and the disulfiram and copper gluconate combination when compared with the control (Table 8 and Table 9).
Effect of low, medium and high dose disulfiram, copper gluconate and their combination on creatinine levels were significantly increased (p < 0.05) when compared with the control (Tables 10-12).Serum creatinine is an important indicator of renal health because it is an easily measured by-product of muscle metabolism that is excreted unchanged by the kidneys.Plasma creatinine concentration is the most widely used measure for estimation of the glomerular filtration rate (GFR) [23].Creatinine is synthesized primarily in the liver from the methylation of glycocyamine (guanidino acetate, synthesized in the kidney from the amino acids arginine and glycine) by S-adenosyl methionine and is removed from the blood chiefly by the kidneys, via glomerular filtration and proximal tubular secretion.A rise in blood creatinine level is observed only with marked damage to functioning nephrons.A persistent rise in both urea and creatinine is a sign of kidney failure.Kidney failure and death can occur with as little as 1 gram of copper sulphate [24].Heavy metals such as cadmium (Cd), mercury (Hg), lead   Results are expressed as mean ± SEM, the superscript (*) means significant difference with respect to control at p < 0.05 (ANOVA).(Pb), chromium (Cr) and platinum (Pt) are a major environmental and occupational hazard.Unfortunately, these non-essential elements are toxic at very low doses and non-biodegradable with a very long biological half-life.Thus, exposure to heavy metals is potentially harmful.Because of its ability to reabsorb and accumulate divalent metals, the kidney is the first target organ of heavy metal toxicity.The extent of renal damage by heavy metals depends on the nature, the dose, route and duration of exposure.Both acute and chronic intoxication have been demonstrated to cause nephropathies, with various levels of severity ranging from tubular dysfunctions like acquired Fanconi syndrome to severe renal failure leading occasionally to death [17].

Conclusion
Disulfiram and copper gluconate as single agents are nephrotoxic as seen from our results.Administered as a combination, disulfiram/copper gluconate was nephrotoxic as shown by the development of hyperkalemia, hypernatremia, uraemia and increased creatinine levels which were higher for the drug combination when compared to the single agent.pointers to the development of kidney damage in the experimental animals.
The researchers believe that the nephrotoxicity observed is achieved via a synergistic toxicological effect when disulfiram and copper gluconate are combined.These effects were low, medium and high doses and should therefore be used with extreme caution.
Results are expressed as mean ± SEM, the superscript (*) means significant difference with respect to control at p < 0.05 (ANOVA).