Effect of diethyldithiocarbamate in cyclophosphamide-induced nephrotoxicity: Immunohistochemical study of superoxide dismutase 1 in rat.

OBJECTIVES: To investigate the role of diethyldithiocarbamate (DEDTC) in cyclophosphamide (CP)-induced nephrotoxicity in Sprague-Dawley rat. DEDTC is a known chelating agent for copper and zinc. It is also used as a thiol protecting agent, as nuclear factor kappa-light-chain-enhancer of activated B-cells inhibitor and nitric oxide synthase inhibitor. It is also reported to inhibit superoxide dismutase (SOD) both in vitro and in vivo conditions. Considering this wide range of actions, current study investigated the role of DEDTC in CP-induced nephrotoxicity in experimental rat model.
MATERIALS AND METHODS: Thirty-two male rats were randomized into four groups. Group 1, control received only saline ip; Group 2 and 4, received CP at the dose of 150 mg/kg body weight ip on the 4th day, while Group 3 and 4, received DEDTC at the dose of 250 mg/kg alternatively (fractionated dose of 1000 mg/kg). All the experimental animals were sacrificed on the 7th day and organs of interest were collected for biochemical, histopathological, DNA damage, and immunohistochemical assessments.
RESULTS: DEDTC administration was found to further exacerbate the condition of CP-induced kidney damage as assessed by several biochemical and histological parameters. Further, the damage was also significantly reflected in the bladder in DEDTC-treated animals as compared to controls. SOD1 (Cu/Zn- dependent enzyme) expression was found to be decreased and this might be due to the action of DEDTC on SOD and other antioxidants.
CONCLUSION: The present study indicates that DEDTC administration further exacerbated the CP-induced kidney damage in rat.

Introduction

Cyclophosphamide (CP) is one of the most widely used anticancer drugs, which acts by alkylation of DNA.[1] CP-induced toxicities are well characterized in several experimental studies.[234] CP-induced uro- and nephrotoxicites are well characterized.[24] CP-induced renal damage generally remains undiagnosed/or neglected, as a reliable plasma marker for the glomerular damage like plasma creatinine remains unaltered.[45] Several of the mechanisms reported earlier to these aspects of toxicity from our laboratory.[67] However, the exact mechanism for CP-induced nephrotoxicity is unknown, though, potential causative factors for CP-induced nephrotoxicity can be summarized as follows: (i) generation of reactive oxygen and nitrogen species, (ii) decreased lysosomal enzyme activity, (iii) metabolic activation of CP by CYP450, and (iv) poly (ADP-ribose) polymerase activation.[89] Zinc (Zn) plays a very vital role in the maintenance of the normal cellular homeostasis. Zn supplementation has been reported to prevent toxicant-induced nephrotoxicity in animal studies. Zn mediates it protective effects mainly through; (i) restoration of free-radial-antioxidant balance, (ii) upregulation of metallothionein, (iii) upregulation of nuclear factor (erythroid-derived 2)-like 2 (nrf-2).[1011] Diethyldithiocarbamate (DEDTC) is an active metabolite of clinically used drug disulfiram.[12] DEDTC is also widely used for its various types of mechanism of actions such as nuclear factor kappa-light-chain-enhancer of activated B-cells inhibition, superoxide dismutase (SOD1) inhibition, nitric oxide synthase inhibition, and also as thiol protectant.[131415] The low dose (100 mg/kg) was found to be beneficial in protecting toxicant-induced damage.[12] It is also widely used as an experimental chelator at the doses ranging from 500 to 1000 mg/kg.[1617] CP produced oxidative stress is one of the main mechanisms through which it induced damages in various organs. Cu/Zn plays a vital role in maintaining the antioxidant balance in multicellular systems.[18] Earlier, we have reported that Zn plays a vital role in CP-induced testicular toxicity and the decreased Zn levels contributed toward the induction of CP toxicity.[3] Zn was also used as a protectant in CP-induced urotoxicity, which also signifies that Zn plays a crucial role in CP-induced damages.[19] SOD1 was reported to be predominant isoform of SOD in kidney, and several studies have reported the importance of SOD1 in renal damage.[2021] Recently, DEDTC was also shown to aggravate methotrexate-induced testicular damage in rat by inhibiting SOD.[22] A study conducted by Yang et al. has shown the beneficial effects of Zn supplementation on diabetes-induced kidney damage by the upregulation of Nrf2 and its downstream factors such as SOD1 and SOD2.[23] Chelation of Cu/Zn and Cu/Zn-dependent SOD inhibition may be harmful if not characterized properly. Here, an attempt has been made to further characterize the CP-induced toxicity in the presence of DEDTC in Sprague–Dawley (SD) rat.

Materials and Methods

Animals

Experimental protocol of animal study was approved by the Institutional Animal Ethics Committee (IAEC) and experiments were performed on male SD rats (250–270 g, 8–10 weeks). Animals were procured from the Central Animal Facility of the institute in accordance with the committee for the purpose of control and supervision of experimentation on animals guidelines (IAEC 13/30, 16/10-R). All the animals were maintained in the controlled environment such as room temperature (22±2°C); Humidity (50±10%); light (cycle of 12 h light and 12 h dark). Feed and water were given ad libitum and animals were acclimatized for 3 days before the commencement of the experiment.

Chemicals

CP (Endoxan®) was procured from Zydus Cadila and DEDTC as sodium salt (CAS No. 20264-25-3) was purchased from Loba Chemie Pvt. Ltd., Mumbai, India. Until unless mentioned, all the chemicals and reagents were procured from Sigma-Aldrich chemicals, Saint Louis, MO, USA, while primary and secondary antibodies were procured from Santa Cruz Biotechnology, CA, USA.

Experimental design and animal treatment

Thirty-two male rats were randomized into four groups. Group 1, control (CON) received only saline ip; Group 2 (CP) and 4 (CP + DEDTC), received CP at the dose of 150 mg/kg ip once on the 4th day, while Group 3 (DEDTC) and 4 (CP + DEDTC) received DEDTC at the dose of 250 mg/kg ip (4 times) on every alternative day, based on the earlier studies.[24] All the animals were sacrificed on the 7th day and organs of interest were collected for biochemical, histopathological, DNA damage, and immunohistochemical assessments. Sodium DEDTC and CP were dissolved in normal saline.

Biochemical analyses

Oxidative stress parameters, malondialdehyde (MDA) and glutathione (GSH) were estimated as described previously[25] in the kidney tissue, while creatinine, urea, albumin, and alkaline phosphatase (ALP) were estimated enzymatically in plasma by commercially available kits (ACCUREX, Mumbai, India) in accordance with manufacturer's instructions.

Serum and tissue Zn analysis

Serum Zn was directly estimated in the serum as previously described using graphite furnace-atomic absorption spectrometry (GF-AAS) (Analytic Jena, Germany) at 219.3 nm as described by Maremanda et al.[25] Tissue Zn was estimated as previously described,[25] briefly preweighed kidney tissue was digested in nitric acid overnight, centrifuged, and diluted with distilled water for the estimation of Zn using GF-AAS.

Histological evaluation and quantification

Slides for histology were prepared as described earlier.25 Briefly, kidney and bladder tissues were fixed in 10% neutral buffer formalin followed by gradual dehydration in ethanol and xylene. Further, these tissues were embedded in paraffin and 5 μm thin sections were taken for histopathological and immunohistochemical analysis. The rehydrated sections were stained by H&E and periodic acid-Schiff staining (Sigma, USA), followed by mounting with DPX and were examined under the microscope (Olympus BX51, Tokyo, Japan). Alterations in histology such as glomerular volume, cross-sectional area, capsular space, and cytotoxicity were assessed and quantified using Image J software as described by Khan et al.[26] Further, fibrosis was analyzed using picrosirius red and Masson's trichome staining of kidney.[26]

Assessment of DNA damage using alkaline comet assay

A small piece of kidney was treated with 1 ml cold Hank's Balanced Salt Solution containing 20 mM EDTA and 10% dimethyl sulfoxide. The tissue was minced to get single cell suspension and the comet assay was performed as described by Tripathi and Jena.[7]

Immunohistochemistry of superoxide dismutase 1 in kidney

Immunohistochemistry was performed using commercially available kit, Novolink Polymer Detection System (Leica, Milton Keynes, UK) as per manufacturer's instructions using the primary antibodies against SOD1 (Cat No. sc-11407). Briefly, after deparaffinisation and rehydration, sections of kidney (5μm) were taken on the precoated poly-L-lysine slides, incubated in 0.01 M citrate buffer (pH 6.0) at 95°C for 20-30 minutes for antigen retrieval. Further, processing was done according to the manufacturer's instructions, nucleus was stained using hematoxylin and tissue was finally mounted on DPX.

Statistical analyses

Results were calculated as mean ± standard deviation (SD) for each group and were analyzed using GraphPad Prism (V.7, GraphPad Software, CA, USA) statistical software. For calculation of significance difference between multiple groups, one-way analysis of variance was used and post hoc analysis was performed with Tukey's test and P < 0.05 was considered to be statistically significant.

Results

Effect of cyclophosphamide and diethyldithiocarbamate on the body and organ weights

There was no significant change in the body weights observed at the end of the study in any of the groups [Figure 1a]. Relative kidney weight remained unchanged [Figure 1b]. On the contrary, there was a significant increase in the relative bladder weight in CP-treated groups [Figure 1c].

Figure 1

Morphometric and biochemical parameters. (a) Final body weight. (b and c) Organ to body weight ratio of the kidney and urinary bladder (d-k) malondialdehyde, glutathione (reduced), serum alkaline phosphatase, serum albumin, serum blood urea nitrogen, serum creatinine, serum Zn, and kidney Zn were measured at the end of the study. All the values are shown as mean ± standard deviation, n = 3–6 “a” versus CON and “b” versus cyclophosphamide

Effect of cyclophosphamide and diethyldithiocarbamate on biochemical parameters

Although there was an increase in the MDA levels in the CP-treated groups, the increase was found to be nonsignificant. GSH levels were also unaffected by the treatments [Figure 1d and e].

Significant decrease in ALP (a Zn-dependent enzyme) was found in the CP + DEDTC group [Figure 1f]. Albumin was also found to be decreased in the CP and CP + DEDTC groups [Figure 1g]. Further, the level of blood urea nitrogen (BUN) was significantly increased in the CP + DEDTC group [Figure 1h]. Serum creatinine was found to be unaltered [Figure 1i]. Serum Zn was found to be significantly increased in the DEDTC-treated groups, whereas there was no significant change in the kidney Zn levels [Figure 1j and k].

Histological and histomorphometrical analyses of kidney

Histopathological evaluation of rat kidney revealed the nephrotic damage induced by CP as characterized by luminar fragmentation and shedding of tubular epithelium. Further observations showed the vacuolar degeneration of tubular epithelial cells and glomeruli atrophy in CP-treated groups [Figure 2a]. Furthermore, groups treated with CP showed marked increase in tubular damage and glomerular atrophy as compared to control. There was significant increase in capsular space in the DEDTC-treated group [Figure 2]. PAS staining has shown tubular damage [Figure 2a] and deformed glomeruli with significant increase in the capsular space as seen in CP + DEDTC group [Figure 2b]. Further, PAS staining showed that treatment with per se CP significantly decreased the glomerular area and volume in comparison to control [Figure 2].

Figure 2

(a) Periodic acid–Schiff-stained images of the rat kidney showing glomerulus under ×100 magnification along with quantification of capsular space, glomerular area, and glomerular volume. The arrow indicates increase in capsular space. (b) Periodic acid–Schiff-stained images of renal tubules under ×100 magnification showing various types of damages indicated by the arrows. All the values are shown as mean ± standard deviation, n = 4–5 “a” versus CON and “b” versus cyclophosphamide

Picrosirius red and Masson's trichome stain

Sections of rat kidney were stained with Sirius red [Figure 3a] and Masson's trichome [Figure 3b] for the evaluation of fibrosis. There was significant increase in the percent fibrotic area stained red, in the groups treated with CP, DEDTC, and CP + DEDTC as compared to control. Masson's trichome stain was also performed to assess the fibrosis and was found to be significantly increased (in blue), in groups treated with CP, DEDTC, and CP + DEDTC as compared to control. Further, to assess the intensity of damage caused by the treatments, histopathological evaluation of the urinary bladder was carried out [Figure 4], as the dose of CP used in the study also damages the bladder and causes hemorrhagic cystitis. Histopathological evaluation indicates an increase in the urothelial thickness in animals treated with CP. Treatment with CP also showed the presence of inflammatory cell infiltrations, which has been indicated by dotted arrow in Figure 4 and hemorrhage by thin arrow in Figure 4. Treatment with DEDTC further increased the urothelial thickness as compared to control.

Figure 3

(a) Picrosirius red-stained images of the rat kidney showing glomerular fibrosis under ×100 magnification along with quantification. (b) Masson's trichome-stained images of rat kidney showing fibrosis under ×100 magnification along with quantification. All the values are shown as mean ± standard deviation, n = 4–5 “a” versus CON and “b” versus cyclophosphamide

Figure 4

H and E-stained urinary bladder images under ×100 magnification showing increased urothelial thickness, hemorrhage (thin arrow), desquamated epithelial cells (thick arrow), and inflammatory cell infiltrations (dotted arrow) with quantification. All the values are shown as mean ± standard deviation, n = 5; “a” versus CON and “b” versus cyclophosphamide

Assessment of DNA damage in the kidney tissue by alkaline comet assay

There was significant increase in the %DNA damage as compared to control in groups treated with CP and CP + DEDTC [Figure 5]. Further, treatment with DEDTC did not increase the damage.

Figure 5

Alkaline comet assay indicating the DNA damage along with quantification. All the values are shown as mean ± standard deviation, n = 3–5 “a” versus CON and “b” versus cyclophosphamide

Immunohistochemical analysis of superoxide dismutase 1 in kidney and urinary bladder

SOD1 which is a Cu/Zn- dependent enzyme, was found to be significantly decreased in the kidney of animals treated with DEDTC [Figure 6a]. Similarly, there was a declining trend in the SOD1 in case of bladder as observed in kidney but was not statically significant [Figure 6b].

Figure 6

Immunohistochemical staining of superoxide dismutase 1. (a) Hemtoxylin-DAB-stained kidney images under ×100 magnification along with quantification for DAB-positive area. (b) Hemtoxylin-DAB-stained urinary bladder under ×100 magnification along with quantification for DAB-positive area. All the values are shown as mean ± standard deviation, n = 4–5. “a” versus CON and “b” versus cyclophosphamide

Discussion

Treatment with CP induced nephrotoxicity and urotoxicity in rat, as evident by various parameters used in the present investigation. It has been reported that CP-induced nephrotoxicity and urotoxicity are mainly due to its metabolite acrolein.[2728] CP-induced damage was reflected in the relative organ weight of bladder, but not in the kidney, and this was in agreement with earlier findings carried out with CP to produce nephro- and urotoxicity.[29] MDA levels, which indicate the lipid peroxidation, was found to be on a higher side in CP and CP + DEDTC treated animals, but this was found to be nonsignificant. GSH which generally protects against various toxic insults was not significantly altered in the present treatment regimens. The probable reason for this could be that the organ may produce excess GSH or antioxidants to protect against the toxic insults, which was suggested in our earlier report that short-term treatment with CP in mice led to an increase in the protective enzymes or phase II detoxifying enzymes.[7] Initiation of kidney damage was confirmed by altered levels of ALP, albumin, and BUN. However, creatinine level remained unaltered. Serum Zn levels were significantly increased in the DEDTC-treated groups; this might be due to the chelation effect of DEDTC and the compromised kidney function which was unable to clear out the DEDTC-Zn complex.

Histopathological evaluation with H and E and PAS staining suggested that there was an increase in capsular space, decrease in glomerular area as well as volume, indicating the tubular damage. The similar findings were also reflected in picrosirius red and Masson's trichome staining, which specifically stained the collagen suggesting that there was an increase in the fibrotic area by the CP and CP + DEDTC treatment. The damage was also reflected in the bladder and a significant increase was observed in the thickness of urothelium in the CP + DEDTC treated groups as compared to CP and other groups. There was also an increase in the DNA damage by CP treatment, DEDTC has no further exaggerative effects on CP-induced DNA damage. This suggests that DEDTC-induced damage in the present study might be due to other mechanisms and did not involve DNA damage. To further characterize, immunohistochemical study with SOD1 was carried out. The results of SOD1 immunohistochemical study suggested that DEDTC significantly decreased the SOD1 levels, in the kidney suggesting that DEDTC showed its toxic effects by Cu/Zn chelation and inhibition of SOD1. This further strengthens the claim that DEDTC inhibits SOD as reported earlier.[15] Recent study claims that DEDTC can act as a pro-oxidant in zebrafish, which further supports the notion that DEDTC affects the antioxidant balance in animals.[30]

Conclusion

The present findings suggest that Cu/Zn SOD plays an important role in CP-induced nephrotoxicity, supported by its urotoxic findings. Thus, further studies involving specific chelators of Zn such as TPEN and inhibitors of SOD1 along with CP are suggested to understand the exact role played by these antioxidant systems in CP-induced toxic insults.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.