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Abstract

Earlier we have shown that exposure to copper-nitrilotriacetate (Cu-NTA) manifests toxicity by generating oxidative stress and potent induction of proliferative reaction in the liver and kidney. In the study, we look at the impact of nitroglycerin (GTN) administration on Cu-NTA-induced oxidative stress and hyperproliferative response in the liver and kidney. GTN administration intraperitoneally to male Wistar rats after Cu-NTA administration intraperitoneally caused substantial protection against Cu-NTA-induced tissue injury, oxidative stress and hyperproliferative response. Cu-NTA administration at a dose of 4.5 mg/kg body weight produces significant (p < .001) elevation in biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (CREA) with a concomitant increase in microsomal lipid peroxidation. Along with these alterations, we discovered a substantial increment in [³H]thymidine incorporation into hepatic and renal DNA synthesis (p < .001). Cu-NTA-induced tissue damage and lipid peroxidation in hepatic and renal tissues were inhibited by GTN treatment in a dose-dependent manner (p < .05–0.001). Furthermore, GTN can suppress the hyperproliferative response elicited by Cu-NTA by down-regulating the rate of [³H]thymidine incorporation into hepatic and renal DNA (p < .01–0.001). Protective effect of GTN against Cu-NTA was also confirmed by histopathological changes in liver and kidney. This result suggests that GTN may serve as a scavenger for reactive oxygen species (ROS) and reduces toxic metabolites of Cu-NTA, thereby avoiding tissue injury and oxidative stress. Further, administration of NO inhibitor, NG-Nitroarginine methyl ester (L-NAME), exacerbated Cu-NTA induced oxidative tissue damage and cell proliferation. Overall, GTN reduces Cu-NTA-induced tissue damage, oxidative stress, and proliferative response in the rat liver and kidney, according to these findings. On the basis of the above results, present study suggests that GTN may be a potential therapeutic agent for restoration of oxidative damage and proliferation to liver and kidney.

Keywords

oxidative stress nitric oxide glyceryl trinitrate hepatic and renal protection

Introduction

Copper is an essential trace element that plays an important role in different biological processes. The copper entering the liver may be stored in hepatic metallothionein and released into the plasma in ceruloplasmin or secreted in the bile later.^1–4 The kidney can contribute to copper homeostasis by sequestration of copper bound to metallothionein and excretion of the metal as copper-metallothionein, therefore, the homeostasis of this metal ion must be under exquisite regulatory control.⁵ Copper is redox-active, causing oxidative stress, DNA damage, and cell injury by catalyzing the formation of hydroxyl radicals in a Fenton-like process.^6–9 Previous studies have shown that copper overload overwhelms copper transport and liver storage capacity, leading to oxidative damage with enhanced lipid peroxidation, apoptosis and liver dysfunction.^10–13 When hepatocytes have excess copper, then it is released into the blood and is accumulated in the kidney, thus, degenerative changes have been observed in the proximal convoluted tubules of kidneys accompanying the hepatic changes.¹⁴ Copper has been shown to cause DNA damage and cell apoptosis that has been explained based on the copper binding to the N-7 guanine residue of DNA and generating reactive oxygen species (ROS) through the oxidation-reduction reaction.^15,16

Many epidemiological studies have investigated the relationship between exposure to excess copper environmentally and occupationally and adverse health effects.^3,6,12,13 Nitrilotriacetate is an amino tricarboxylic acid that forms water-soluble chelate complexes with a variety of metal cations.¹⁷ It has been used as a chelating agent in many industrial, domestic and agricultural applications due to its ability to complex metals. Although it is biodegradable, its usage is controversial because of its organ toxicity.¹⁸ In artificial nucleases, NTA as a metal-chelator cleaves DNA primarily by metal-bound ROS, and therefore, incorporation of these metal complexes in artificial nucleases has shown to provide more controlled delivery of ROS to targeted genes.^7,19 Copper forms water-soluble and chemically stable chelate with NTA at a neutral pH, thereby its toxicity, assimilation and accumulation in organisms changed to a greater extent.²⁰ The exposure of copper take place in occupational settings of the metallurgic industry via effluent in soil and water where it may occasionally be found in the chelated form with nitrilotriacetate (NTA), as a part of dental amalgams, dental casting alloys, and some intrauterine contraceptive devices (IUDs).^6,7,17,18 It has been reported that copper is gradually and constantly introduced as a corrosion product of IUDs, causing increment in 8-OH-dG and mitotic indices in hepatic and renal tissues of rats, and implying that the corrosion products of copper implants may be harmful to many tissues.²⁰ This necessitates a risk evaluation of copper-related hazards.^15,21,22

Nitric oxide (NO) an endogenous derived free radical performs several important physiological functions.^23,24 It has been demonstrated that NO affords protection against several toxicants and also acts as an intracellular antioxidant.^25–28 The oxidative stress and reduced NO bioavailability impair the vascular protective function of the endothelium.²⁹ The specific role of NO, on the other hand, is still debated because it may both enhance and prevent tissue and cellular damage in different situations. NO has been demonstrated to damage DNA and is a powerful endogenous carcinogen.³⁰ However, it has also been shown to protect cells from apoptosis and may attenuate hyperproliferative response and necrosis in experimental animals.^31–34 Thus, the type of insult, the amount and source of NO generation, the abundance of reactive oxygen species (ROS) as well as specific cell type and the cellular redox status, are likely to affect the action of NO.

ROS are known to be pathophysiological mediator of many disease conditions. The oxidative stress induced by well-known toxins including ferric nitrilotriacetate (Fe-NTA) and Cu-NTA has been attributed to cause tissue injury in liver and kidney.^34,35 Previously we have shown that exposure to Cu-NTA manifests toxicity by generating oxidative stress and potent induction of proliferative reaction in the liver and kidney³⁵ and glyceryl trinitrate (GTN), a nitric oxide donor, abrogates Fe-NTA-induced oxidative stress and renal damage.³⁴ Based on previous findings and considering NO’s dual activity as both a cytotoxic and a cytoprotective mediator, we chose to investigate the effects of NO exposure applying GTN, traditionally employed as a vasodilator, on Cu-NTA-induced hepatic and renal histopathological changes, oxidative stress and hyper proliferative response in rats.

Materials and methods

Chemicals

Nitrilotriacetic acid (NTA), thiobarbituric acid (TBA), 2,4-dinitrophenylhydrazine (DNPH), N^G-nitro-l-arginine methyl ester (L-NAME), glyceryl trinitrate (GTN), alanine, aspartate, tris buffer, sodium tungstate and diacetylmonoxime were purchased from Sigma-Aldrich (St Louis, MO, USA). Perchloric acid (PCA), ferric nitrate, sodium chloride, potassium chloride, trichloroacetic acid (TCA) and sodium hydroxide were obtained from CDH, India. [³H]Thymidine (specific activity 82 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK). All other chemicals used in this study were of the highest purity grade available commercially.

Preparation of Cu-NTA

Sodium bicarbonate was used to adjust the pH to 7.4 after mixing copper sulfate solution with NTA (disodium salt) solution in a 1:4 M ratio.³⁵

Animals

Animals’ experiments were authorized by Jamia Hamdard animal care committee, animal care and handling followed Indian Council of Medical Research regulations (JH AEC-03/1997). The study employed male albino rats of the Wistar strain (125–150 g weighing and 4–6 weeks old) from Jamia Hamdard Central Animal House Colony. The rats were kept in polypropylene cages in a group of 12 in a room that was kept at 22 ± 2°C with a 12-h light/dark cycle. They were fed ad libitum tap water and normal laboratory feed (Hindustan Lever Ltd, Bombay, India). After a week of acclimation, the rats were utilized.

Animal experimental design

For biochemical, serum and histopathological studies, 42 male Wistar rats were taken and randomly divided into seven groups (six rats in each group). Intraperitoneal injections were given to all of the animals and they were sacrificed by cervical dislocation at 12 h after Cu-NTA administration. The dose selection and route of administration for GTN, N^G-nitro-L-arginine methyl ester (L-NAME) and Cu-NTA were based on our previous published studies.^33–35 Treatment groups are shown as below:

Group I received intraperitoneal injection of saline (10 mL/kg body weight).

Group II received intraperitoneal injection of GTN (6 mg/kg body weight, 1 h before killing the rats).

Group III received intraperitoneal injection of L-NAME (40 mg/kg body weight).

Group IV received intraperitoneal injection of Cu-NTA (4.5 mg/kg body weight).

Group V received intraperitoneal injections of Cu-NTA (4.5 mg/kg body weight) and GTN (3 mg/kg body weight, 1 h after Cu-NTA administration).

Group VI received intraperitoneal injections of Cu-NTA (4.5 mg/kg body weight) and GTN (6 mg/kg body weight, 1 h after Cu-NTA administration).

Group VII received intraperitoneal injections of Cu-NTA (4.5 mg/kg body weight) and L-NAME (40 mg/kg body weight, 1 h after Cu-NTA administration).

There was no mortality in each group. The animals were sacrificed by cervical dislocation. Just before the killing, blood was obtained from these animals through retro ocular sinus puncture for serum separation. The liver and kidneys were quickly removed, rinsed and immediately processed for the lipid peroxidation assay. Serum was separated and utilized to study hepatic and renal damage markers.

To study [³H]thymidine incorporation into hepatic and renal DNA, there were additional 42 male Wistar rats were used. The same treatment protocol and dose regimen was followed as described above. In addition, each rat was administered [³H]thymidine (30 μCi/animal/0.2 mL saline) 2 h before the killing. Their liver and kidneys were quickly removed, rinsed with ice-cold saline thoroughly and processed to study [³H]thymidine incorporation into hepatic and renal DNA.

[³H]Thymidine incorporation assay

The liver and kidneys were promptly removed, washed and 1 G of liver and kidney were homogenized in ice-cold water (10% w/v). The precipitate was incubated overnight at 4°C with cold PCA (10%) after rinsing with cold TCA (5%). It was centrifuged after incubation, and the precipitate was rinsed with 5% of cold PCA. All centrifugations had been done at 4°C up to this point. The precipitate was dissolved in warm PCA (10%), incubated for 30 min in a boiling water bath and then filtered using Whatman 50 filter paper. By adding the scintillation fluid to the filtrate, it was employed for [³H]thymidine incorporation counting in a liquid scintillation counter. DPM/g DNA was used to calculate the quantity of [³H]thymidine incorporated.^33,34

Preparation of microsomes

The liver and kidneys were rapidly removed and 1 G of liver and kidney were homogenized in chilled phosphate buffer (0.1 m, pH 7.4) containing potassium chloride (KCl; 1.17% w/v) using a homogenizer before being perfused with ice-cold saline (0.85% w/v sodium chloride). To remove the nuclear debris, the homogenate was filtered through a muslin cloth and centrifuged (5 min at 800 g). The aliquot obtained was centrifuged (20 min at 105,000 g) to get post-mitochondrial supernatant (PMS), which was ultra-centrifuged, and the pellet (microsomal fraction) was suspended in phosphate buffer.

Lipid peroxidation

The reaction mixture comprised 0.58 mL phosphate buffer (0.1 m, pH 7.4), 0.2 mL microsome (10% w/v), 0.2 mL ascorbic acid (100 mm), and 0.02 mL ferric chloride in a total volume of 1.0 mL (100 mm). For 1 h, the reaction mixture was incubated in a shaking water bath at 37°C. The reaction was halted by adding 1.0 mL trichloroacetic acid (TCA 10% w/v) to the mixture. After adding 1.0 mL TBA (0.67% w/v), all tubes were immersed in a boiling water bath for 20 min. Finally, the tubes were placed in a cold bath and centrifuged for 10 min at 2500 g. Quantification of malondialdehyde (MDA) was performed spectrophotometrically at 535 nm by comparing the optical density of the supernatant with a blank reagent.³⁴

Serum transaminases activity

Around 0.5 mL substrate, either α l-alanine (200 mm) or l-aspartate (200 mm) with 2 mm α-ketoglutarate) was incubated in a water bath (at 37°C) for 5 min. The volume was then adjusted to 1 mL with sodium phosphate buffer after the serum (0.1 mL) addition. For GPT and GOT, the reaction mixture was incubated for exactly 30 and 60 min. After that, 0.5 mL of 2, 4-dinitrophenyl hydrazine (1 mm) was introduced to the reaction mixture and allowed for another 30 min. In the end, the color was generated by the exposure of 5 mL NaOH (0.4 N) and the final product was analyzed at 505 nm.³⁶

Estimation of creatinine

Around 1.0 mL distilled water, 1.0 mL sulfuric acid (0.6 N) and 1.0 mL sodium tungstate (5%), and were added to 1.0 mL plasma/serum. The mixture was centrifuged for 5 min at 800 g and the obtained supernatant was added to the mixture containing 1.0 mL sodium hydroxide (0.75 N) and 1.0 mL picric acid (1.05%). After exactly 20 min, the absorbance at 520 nm was measured.³⁷

Estimation of blood urea nitrogen

To 0.5 mL of protein-free filtrate were added 3.5 mL distilled water, 0.8 mL diacetylmonoxime (2%), and 3.2 mL sulfuric acid-phosphoric acid reagent (the reagent was prepared by mixing 150 mL of 85% phosphoric acid with 140 mL water and 50 mL conc. H₂SO₄). After 30 min in a boiling water bath, the reaction mixture was cooled and at 480 nm, the absorbance was measured.³⁷

Histopathological studies

Sections of liver and kidney tissues from each group was fixed with 10% neutral buffered formalin solution, then followed by embedding in paraffin for microtome sectioning 5–6 μm thick sections. Afterwards, the resultant sections were stained with hematoxylin–eosin (H&E), and finally histopathological changes were captured in a high-resolution light microscope.

Statistical analysis

Significant differences between groups were investigated using a one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. All values were highlighted as mean ± standard error of the mean (SEM). A p values <.05 were considered as significant.

Results

Determination of Cu-NTA-induced lipid peroxidation and the role of GTN

The effect of GTN administration on Cu-NTA-mediated enhancement in hepatic and renal lipid peroxidation is shown in Table 1. Cu-NTA administration resulted in a significant (p < .001) increase in lipid peroxidation of the membrane in both liver and kidney. The administration of GTN at dose levels of 3 mg/kg body weight, resulted in a significant inhibition (p < .05) of Cu-NTA induced formation of TBA reacting species in the kidney when compared to Cu-NTA alone treated group. However, at a higher dose of GTN at 6 mg/kg body weight, the inhibition of Cu-NTA induced-lipid peroxidation was significantly (p < .001) observed in the liver and kidney when compared to Cu-NTA alone treatment group. L-NAME significantly (p < .05) enhanced the peroxidation of membrane lipids in the liver and kidney of Cu-NTA treated rats as compared to the Cu-NTA treated group.

Table 1.

The effect of GTN administration on Cu-NTA-induced hepatic and renal lipid peroxidation.

Treatment groups	Hepatic lipid peroxidation (nmol MDA formed/h/g of protein)	Renal lipid peroxidation (nmol MDA formed/h/g of protein)
Group I- saline	10.65 ± 0.91	15.34 ± 1.13
Group II- GTN	9.32 ± 1.24	17.28 ± 1.72
Group III-L-NAME	12.81 ± 1.68	20.93 ± 1.89
Group IV- Cu-NTA	25.46 ± 1.77^†	38.70 ± 2.51^†
Group V-Cu-NTA + GTN (dose 1)	21.59 ± 1.85**^†	32.27 ± 1.34**^†
Group VI-Cu-NTA + GTN (dose 2)	16.37 ± 0.87***^†	25.18 ± 1.42***^†
Group VII-Cu-NTA + L-NAME	33.61 ± 2.06**^†	45.74 ± 1.78**^†

Data represent mean ± SEM of six rats/group. Saline treated group (I) serves as control for groups IV, V, VI and VII (†p < .001). Significantly (†p < 0.001) different from saline-treated group. Cu-NTA treated group (IV) serves as control for groups V, VI and VII (**p < .05 and ***p < .001). Significantly (**p < .05 and ***p < .001) different from saline-treated group. Dose regimen and treatment protocol are described in the text. GTN: Glyceryl trinitrate; L-NAME: NG-nitro-L-arginine methyl ester; Cu-NTA: Cupric nitrilotriacetate; Dose 1: 3 mg/kg body weight of GTN; Dose 2: 6 mg/kg body weight of GTN.

Role of GTN on Cu-NTA-mediated enhancement in the value of serum creatinine and blood urea nitrogen.

Cu-NTA administration led to significant (p < .001) enhancement in the levels of serum creatinine and blood urea nitrogen, markers of kidney damage, respectively. Treatment of GTN to rats receiving Cu-NTA led to a considerable decrease in creatinine and blood urea nitrogen levels. The effect was dependent on the dose of GTN. The changes in levels of BUN and CRN are shown in Table 2. Administration of L-NAME further enhanced the levels of serum creatinine and blood urea nitrogen in Cu-NTA treated group VII as compared to the Cu-NTA group IV.

Table 2.

The effect of GTN administration on biomarkers of kidney function tests, blood urea nitrogen (BUN) and creatinine (CRN) in Cu-NTA treated rats.

Treatment groups	Blood urea nitrogen (IU/L)	Creatinine (IU/L)
Group I- saline	18.51 ± 2.63	1.54 ± 0.25
Group II- GTN	16.84 ± 2.12	1.51 ± 0.21
Group III- L-NAME	20.79 ± 2.87	1.78 ± 0.19
Group IV-Cu-NTA	52.17 ± 3.26^†	3.96 ± 0.43^†
Group V-Cu-NTA + GTN (dose 1)	41.05 ± 2.94**^†	2.71 ± 0.32**^†
Group VI-Cu-NTA + GTN (dose 2)	35.89 ± 3.02*^†	2.03 ± 0.20*^†
Group VII-Cu-NTA + L-NAME	59.42 ± 2.76^†	4.67 ± 0.38^†

Data represent mean ± SEM of six rats/group. Saline treated group (I) serves as control for groups IV, V, VI and VII (†p < .001). Significantly (†p < .001) different from saline-treated group. Cu-NTA treated group (IV) serves as control for groups V, VI and VII (*p < .01 and **p < .05). Significantly (*p < .01 and **p < .05) different from saline-treated group. Dose regimen and treatment protocol are described in the text. GTN: Glyceryl trinitrate; L-NAME: NG-nitro-L-arginine methyl ester; Cu-NTA: Cupric nitrilotriacetate; Dose 1: 3 mg/kg body weight of GTN; Dose 2: 6 mg/kg body weight of GTN.

Determination of hepatic toxicity markers AST and ALT and the role of GTN

In this investigation, the serum samples of Cu-NTA treated rats, AST and ALT activities were substantially increased compared to control group I (p < .001; Figure 1). Treatment of the rats with GTN in Cu-NTA-treated groups volt and VI caused a dose-dependent significant reduction in the levels of AST and ALT as compared to group IV (Cu-NTA treated animals). The activity of AST and ALT was significantly (p < .05) increased with L-NAME administration in Cu-NTA treated group VII as compared to the Cu-NTA group IV.

Figure 1.

The effect of GTN administration on biomarkers of liver function tests, aspartate aminotransferase (AST) and alanine transaminase (ALT) in rats treated with Cu-NTA. (I) Saline, (II) GTN, (III) L-NAME, (IV) Cu-NTA, (V) Cu-NTA + GTN (Dose 1), (VI) Cu-NTA + GTN (Dose 2), (VII) Cu-NTA + L-NAME. Data represent mean ± SEM of six rats/group. Saline (I) treated group serves as control for groups IV, V, VI and VII (†p < .001). Significantly (†p < .001) different from saline-treated group. Cu-NTA treated group (IV) serves as control for groups V, VI and VII (*p < .01, **p < .05 and ***p < .001). Significantly (*p < .01, **p < .05 and ***p < .001) different from saline-treated group. Dose regimen and treatment protocol are described in the text. GTN: Glyceryl trinitrate; L-NAME: NG-nitro-L-arginine methyl ester; Cu-NTA: Cupric nitrilotriacetate; Dose 1: 3 mg/kg body weight of GTN; Dose 2: 6 mg/kg body weight of GTN. Effect of GTN Administration on Cu-NTA-Induced Rate of [³H]Thymidine Incorporation into Hepatic and Renal DNA.

Administration of Cu-NTA at a dose of 4.5 mg/kg body weight, increased (p < .001) in the [³H]thymidine incorporation into hepatic and renal DNA, as compared to their saline-treated controls. Subsequent administration of GTN at doses 3 mg/kg body weight, resulted in a significant (p < .05) reduction in the [³H]thymidine incorporation in the kidney as compared to the Cu-NTA treated control group. At a higher dose of GTN at 6 mg/kg body weight, there was significant amelioration in both hepatic (p < .01) and renal (p < .05) [³H]thymidine incorporation, as compared to Cu-NTA treated group. Administration of NO inhibitor, L-NAME to Cu-NTA-treated animals caused further increase in [³H]thymidine incorporation in DNA of the liver (p < .05) as compared to the corresponding values in Cu-NTA-treated rats as shown in Figure 2.

Figure 2.

The effect of GTN administration on Cu-NTA-mediated induction in the rate of [³H]thymidine incorporation into hepatic and renal DNA. (I) Saline, (II) GTN, (III) L-NAME, (IV) Cu-NTA, (V) Cu-NTA + GTN (Dose 1), (VI) Cu-NTA + GTN (Dose 2), (VII) Cu-NTA + L-NAME. Data represent mean ± SEM of six rats/group. Saline (I) treated group serves as control for groups IV, V, VI and VII (†p < .001). Significantly (†p<0.001) different from saline-treated group. Cu-NTA treated group (IV) serves as control for groups V, VI and VII (*p < .05 and **p < .001). Significantly (*p < .05 and **p < .001) different from saline-treated group. Dose regimen and treatment protocol are described in the text. GTN: Glyceryl trinitrate; L-NAME: NG-nitro-L-arginine methyl ester; Cu-NTA: Cupric nitrilotriacetate; Dose 1: 3 mg/kg body weight of GTN; Dose 2: 6 mg/kg body weight of GTN. Effect of GTN Administration on Cu-NTA-Induced Hepatic Histopathological Changes.

The effect of GTN administration on Cu-NTA induced hepatic histopathological changes is shown in Figure 3. Administration of Cu-NTA caused infiltration of the inflammatory cellular components ( ) and degeneration of hepatocytes ( ) (Figure 3(c)). These changes were restored on subsequent administration of GTN, as evident from mild inflammatory cellular infiltrates and mild degeneration of hepatocytes (Figure 3(d)). L-NAME administration in Cu-NTA-treated rats further enhanced pathological changes showing enhanced number of inflammatory cells and degeneration of hepatocytes (Figure 3(e)). However, GTN administration alone showed no pathological changes as shown in Figure 3(b).

Figure 3.

The effect of GTN administration on Cu-NTA-induced hepatic histopathological changes. Dose regimen and treatment protocol are described in the text. A: Saline (x125), B: GTN (x250), C: Cu-NTA (x500), D: Cu-NTA + GTN (x500), E: Cu-NTA + L-NAME (x500). Infitration of inflammatory cell (yellow arrow) and degeneration of hepatocytes (red arrow). Effect of GTN Administration on Cu-NTA-Induced Renal Histopathological Changes.

The histopathological findings of the effect of GTN on Cu-NTA-induced renal damage is shown in Figure 4. The renal histopathological changes induced by Cu-NTA includes glomerular swelling with obliteration of space in Bowman’s capsule ( ), congested blood vessel ( ) and swelling of tubular cells ( ) (Figure 4(c)). GTN treatment alone at higher dose shows normal histopathology (Figure 4(b)). Subsequent administration of GTN resulted in reversal of Cu-NTA-induced renal histopathological changes which includes normal cellular lining and space in Bowman’s capsule suggesting that GTN has protective effect against Cu-NTA induced damage (Figure 4(d)). In contrast, L-NAME treatment further enhanced Cu-NTA-induced renal histopathological changes (Figure 4(e)). These observations showed that GTN had a potent preventive effect on liver and kidney damage caused by Cu-NTA and could effectively relieve the damage degree of liver and kidney tissue, similar to the findings of biochemical analysis.

Figure 4.

The effect of GTN administration on Cu-NTA-induced renal histopathological changes. Dose regimen and treatment protocol are described in the text. A: Saline (x250), B: GTN (x250), C: Cu-NTA (x250), D: Cu-NTA + GTN (x250), E: Cu-NTA + L-NAME (x250). Bowman’s space obliteration (yellow arrow), congested blood vassel (white arrow) and tubule cells inflammation (red arrow).

Discussion

Present study showed that GTN treatment dose-dependently ameliorated Cu-NTA-induced hepatic and renal tissue injury, oxidative stress and hyperproliferative response, while an acute dose of L-NAME, a NO inhibitor, intensifies the Cu-NTA induced hepatic and renal tissue injury, oxidative stress and hyperproliferative response. The finding suggests that GTN may serve as a scavenger for ROS and reduces toxic metabolites of Cu-NTA, thereby avoiding tissue injury, oxidative stress and hyperproliferative response.

The liver plays a key role in endogenous hormone metabolism and xenobiotic detoxification, is a primary organ affected by NO under diverse liver disease conditions.³⁸ NO plays a wide range of roles in the liver, it can act as both prooxidant and antioxidant, and can both induce and inhibit apoptosis in the liver.^15,38,39 A number of NO donor prodrugs have been created based on the biological significance of NO. Inhibition of Cu-NTA induced oxidative alterations by NO appears to be due to its ability to quench ROS particularly O₂^-, contributing to its protective effect. NO inhibits lipid peroxidation in the current study, which is consistent with previous research which indicates that NO inhibits O₂-dependent, iron (hemoprotein)-catalyzed lipid peroxidation.^19,31,34,40

In the present study, Cu-NTA administration also increased renal lipid peroxidation, resulting in a generational imbalance of oxygen-derived radicals, causing oxidative damage and renal injury, as evident by the increase in serum creatinine and blood urea nitrogen. To clarify the role of nitric oxide, rats were given the nitric oxide donor, GTN at two different doses, 3 and 6 mg/kg body weight, or the nitric oxide synthase inhibitor, L-NAME after Cu-NTA injection. GTN was found to ameliorate the nephrotoxic effect of Cu-NTA whereas L-NAME worsened its toxic effect as evidenced by the increase in nephrotoxicity markers, creatinine and blood urea nitrogen. Our results are in harmony with other renal toxicants induced injury, nitric oxide donor and inhibitor,⁴¹ where the protective effect of NO donor, GTN was demonstrated by a reduction in serum creatinine and blood urea nitrogen concentrations. The increase in creatinine and blood urea nitrogen by Cu-NTA administration relates to poor renal function. Earlier data with NO donor has shown the reversal of renal toxicity by gentamicin in rats with an increase in glomerular filtration rate and decrease in serum creatinine.⁴² This was explained by the increase of NO production, a strong vasodilator providing the improvement of hemodynamics. The impact of nitric oxide synthase inhibition on renal failure has been studied. Previously published research revealed that endothelial NOS restores renal function after damage whereas inducible NOS activation leads to increase NO generation, which promotes tubular cytotoxicity and worsens renal failure.^42,43 In our study, the nephroprotective effects of GTN were reversed by L-NAME which is a non-selective inhibitor. L-NAME has been shown to cause tubular deformation by inhibition of NOS while positive effects of NOS inhibition may be prevented.³¹ Nitric oxide is the most important paracrine modulator and mediator of controlling renal functions such as renal blood flow, renal autoregulation, glomerular filtration, renin excretion and Na excretion. Nitric oxide is involved in diabetic nephropathy, inflammatory glomerular abnormalities, acute and chronic kidney failure, medicine-induced nephrotoxicity, and a variety of kidney diseases.^42,43 Numerous studies of metal-induced lipoprotein oxidation reactions, as well as hepatic, myocardial inflammatory and ischemic-reperfusion damage models also indicated that stimulation of NO production.^42–46 NO exposure suppresses oxidant-related mechanisms and blunts the ultimate expression of molecular or tissue damages.^44–46 The protective effect of GTN, a prodrug generating NO⁴⁷ could possibly be attributed to its ability to scavenge the lipid peroxyl radical, while diminishing the level of oxidative stress.^48,49

Cu-NTA has been shown to exhibit proliferative response as evident from several folds increase in [³H]thymidine incorporation into hepatic and renal DNA.³⁵ It has also been shown a potent tumor promoter in the kidney.³⁵ The observed inhibition of [³H]thymidine incorporation into hepatic and renal DNA, following GTN administration, down-regulate the rate of DNA synthesis owing to the reduction in the generation of reactive toxic intermediates including, 4-hydroxy-2-nonenal (HNE)-modified protein and peroxynitrite formation, which is parallel to the studies showing NO-mediated inhibition of DNA or protein synthesis⁵⁰ and mesangial cell proliferation.⁵¹ Cu-NTA have shown to induce mitotic figures in the liver and kidney suggesting the proliferative and tumor promoting potential of Cu-NTA in both organs.²⁰ Similarly, in Cu-NTA-mediated induction of [³H]thymidine incorporation, administration of NO might has counteracted ROS production, thereby, reducing their oxidant potential as well as conjugating to form a less toxic nitrosyl-metal complex and consequently suppressing Cu-NTA-mediated enhanced rate of DNA synthesis.

Conclusions

In summary, our studies suggest that NO delivery through the administration of GTN could be used to protect against Cu-NTA-mediated hepatic and renal damage and proliferation by inhibiting the tissue oxidative stress, which greatly depend on its amount, location and duration of generation. The quantity and length of NO exposure, the kind of non-NO-related toxic insults, and the diseased condition of the organ are all factors that influence whether NO has helpful or detrimental effects. On the basis of the above results, the present study suggests that GTN may be a potential therapeutic agent for restoration of oxidative damage and proliferation to liver and kidney.

Footnotes

Acknowledgements

This work is taken in part from PhD Thesis of Ayesha Rahman.

Author contributions

A.R.A.; conceptualization, methodology, validation, visualization, analysis, investigation, resources, writing-original draft preparation, S.V.S. and M.I.; writing, reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Ayesha Rahman Ahmed

Senty Vun-Sang

Mohammad Iqbal

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Therapeutic role of nitroglycerin against copper-nitrilotriacetate induced hepatic and renal damage

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Preparation of Cu-NTA

Animals

Animal experimental design

[3H]Thymidine incorporation assay

Preparation of microsomes

Lipid peroxidation

Serum transaminases activity

Estimation of creatinine

Estimation of blood urea nitrogen

Histopathological studies

Statistical analysis

Results

Determination of Cu-NTA-induced lipid peroxidation and the role of GTN

Role of GTN on Cu-NTA-mediated enhancement in the value of serum creatinine and blood urea nitrogen.

Determination of hepatic toxicity markers AST and ALT and the role of GTN

Discussion

Conclusions

Footnotes

Acknowledgements

Author contributions

Declaration of conflicting interests

Funding

ORCID iDs

References

[³H]Thymidine incorporation assay