3.1. Introduction. Deptt. of Med. Elemn. & Toxicology 71 PhD Thesis

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1 3.1. Introduction NTA can make complexes with metal ions such as Fe 3+ or Cu 2+ (Irwing et al., 1966). Fe- NTA, a complex of Fe 3+ and NTA, is a strong nephrotoxic agent and a renal carcinogen. It has been established that an iron-chelate of nitrilotriacetate, Fe-NTA induces acute and sub-acute renal injury in animals (Awai et al., 1979; Hamazaki et al., 1985). Some studies have reported that oxygen free radical was formed from redox-active iron and was detected in the serum of Fe- NTA-treated rats (Liu et al., 1993; Xang et al., 1995). Excess free iron causes free radical-mediated peroxidation of membrane lipids and leads to generation of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) (Jacobs, 1980; Chen at al., 2001). Iron-catalyzed oxidative stress is believed to be the main offender involved in the pathogenesis of iron induced cancer (Toyokuni et al., 2002; Toyokuni, 1996).It is considered that reactive oxygen species (ROS) induced by univalent reduction of molecular oxygen in presence of iron and forms superoxide anion (O 2- ) via Fenton/Haber Weiss or auto-oxidation reaction (Liochev and Fridovich, 2002; Gupta et al., 2009). Fe-NTA is also toxic to liver, acting through the production of ROS that initiates oxidative damage of DNA and lipids (Gupta et al., 2009; Iqbal et al., 2009). There is an emerging interest in the use of naturally occurring phytochemicals with renalprotective and antioxidant activity in Fe-NTA intoxication therapy. Over-whelming evidence from epidemiological, experimental (in vivo and in vitro) and clinical trial data indicates that a plant-based diet can lessen the risk of chronic diseases, particularly cancer (Gupta at al., 2009; Rafter, 2002).Studies have shown that fruits, vegetables, spices, tea, and medicinal herbs rich in antioxidants and other micronutrients protect against diverse forms of chemically induced injuries and carcinogenesis, inhibit DNA-damage, mutagenesis and lipid peroxidation etc. (Sarkar and Bhaduri, 2001).Caffeic acid is a widespread phenolic acid that occurs naturally in many agricultural products such as fruits, vegetables, wine, olive oil, and coffee (Mattila and Kumpulainen, 2002;shahidi and Naczk, 1995). It is a potent antioxidant (Chan and Ho, 1997), metal chelating agent (Psotova et al., 2003), anti-inflammatory (Chao et al., 2010), free radical scavenger (Gulcin, 2006) and anti-diabetic agent (Jung et al., 2006). Caffeic acid has been shown to be an inhibitor of the lipoxygenase enzyme. Deptt. of Med. Elemn. & Toxicology 71 PhD Thesis

2 Therefore, in the light of the above evidence we have tried to explore the possible useful role of caffeic acid on renal toxicity, we examined the protective actions of caffeic acid against oxidative stress, inflammation and hyperproliferation against Fe-NTA induced renal toxicity. We studied the efficacy of caffeic acid against lipid peroxidation, serum toxicity markers (LDH, BUN and Creatinine), the reduced glutathione (GSH) and glutathione dependent enzymes, neutrophil infiltration (MPO),nitric oxide (NO), polyamine synthesis (ODC) and renal DNA synthesis (Thymidine incorporation) Results Caffeic acid pretreatment decreased MDA formation MDA formation was measured to demonstrate the oxidative damage on lipid peroxidation in Fe-NTA induced renal injury of Wistar rats. A significant increase of the MDA formation was found in the Fe-NTA treated group when compared with control. We have observed that pretreatment with caffeic acid at both D1 and D2 leads to the significant (p<0.01 and p<0.001 respectively) restoration of membrane integrity when compared to Fe-NTA treated group (Table 4). There was no significant difference in MDA levels of control and only D2 group Caffeic acid attenuates the serum BUN and creatinine level Protective effect of caffeic acid on serum BUN and creatinine level was observed. Significance change in these parameters was found in the Fe-NTA treated (BUN and creatinine) groups as compared to respective control (p<0.001). Pretreatment with caffeic acid was found significantly effective (BUN, p<0.01and Creatinine, p<0.05) in the normalization of these kidney specific markers when compared to Fe-NTA treated group (Table 3). Caffeic acid alone did not show any significant difference as compared to control Renal GSH contents and antioxidant levels restored by pretreatment Animals when subjected to Fe-NTA treatment caused significant decrease in the activities of all glutathione metabolizing enzymes viz GST (p<0.001), GPx (p<0.001) and Deptt. of Med. Elemn. & Toxicology 72 PhD Thesis

3 GR (p<0.001) when compared with control GST, GR and GPx (Table. 1-2).This depletion of GSH dependant enzymes leads to disruption of other oxidative pathway enzymes like SOD (p<0.001).caffeic acid pretreatment restores the level of all glutathione dependent enzymes significantly in both the doses (D1 & D2). No difference in antioxidant enzyme level was found between only D2 group and control Assay of NADPH: Quinone oxidoreductase Quinone oxidoreductase (QR) reflected significant decrease (p<0.001) in the enzyme activity in renal tissue of Toxicant group when compared with control (Table. 2) Caffeic acid significantly restores the level of quinone oxidoreductase (QR) in both the doses D1 (p<0.05) & D2 (p<0.001).only D2 group showed no significant change as compared to control group Xanthine Oxidase, LDH, γ-glutamyl transpeptidase and H 2 O 2 level restored to normal by pretreatment There was significant enhancement of renal microsomal Xanthine Oxidase (p<0.001), γ- glutamyl transpeptidase (p<0.001), LDH (p<0.001) and H 2 O 2 (p<0.001) levels in Fe NTA treatment group (Table. 2-4). Marked reduction was noted in Treated groups at both the doses in all the enzymes. No significant difference was observed in only D2 group and control group Myloperoxidase activity and nitrite level restored by caffeic acid pretreatment Treatment with Fe-NTA resulted in significant increase in the MPO activity (p<0.001) when compared with control. Both the doses of caffeic acid D1 (p<0.05) and D2 (p<0.001) significantly brought the level MPO to normal (Table 6) Caffeic acid alone did not show any significant difference as compared to control. Similar pattern as above was observed in nitric oxide estimation. (Table 5) Effect on tumour promotion markers: ODC and Thymidine Treatment with Fe-NTA caused significant induction in the ODC (p<0.001) and thymidine (p<0.001) activity as compared with respective control group (Table 5). The pretreatment of rats with caffeic acid at both doses D1 and D2 caused significant Deptt. of Med. Elemn. & Toxicology 73 PhD Thesis

4 inhibition in the elevation of ODC D1 (p<0.01), D2 (p<0.001) and thymidine D1 (p<0.01), D2 (p<0.001) activity as compared with Fe-NTA treated control group Effect of caffeic acid pretreatment on LDH Fe-NTA caused significant increase in LDH level (p<0.001) when compared to control. Caffeic acid at higher dose only D2 ( p<0.01) was effective in bringing back LDH levels to normal. No significant difference was found between only D2 group and control group (Table. 3) Discussion The carcinogenesis is multi-step process and free radicals species are reported to be involved in each of these steps initial step being oxidative stress. In our day-to-day life, we are continuously exposed to a range of chemicals that acts as free radical generators. These free radicals are responsible for number of events lead to diseases including cancer. Toxicity to kidneys following by Fe-NTA exposure is a well known phenomenon (Chen at al., 2001; Wu and Qiu, 2001; Kadkhodaee, 2004). On intraperitoneal administration, Fe-NTA is absorbed into portal vein through mesothelium and passes into circulation via liver (Umemura et al., 1990). Low molecular weight, Fe-NTA is easily filtered through the glomeruli into lumen of renal proximal tubules where Fe 3+ -NTA is reduced to Fe 2+ -NTA by glutathione degradation products cysteine or cysteineglycine (Taso and Curthoys, 1980). Cysteine and cysteineglycine are the anticipated reductants that reduce Fe 3+ -NTA to Fe 2+ -NTA. This results in the production of superoxide radicals (O 2 ) which potentiate the iron-catalyzed Haber Weiss reaction to produce hydroxyl radical (OH ), resulting in the progression of lipid peroxidation and oxidative DNA damage (Guder and Ross, 1984; Aruoma et al., 1989). Fe-NTA exposure elevates the levels of redox active iron, which is known to induce the formation of ROS that can readily attack the cellular molecules. Previously published studies from our lab and others have also reported repeated exposure of Fe-NTA to cause acute and sub-acute renal proximal tubular damage and subsequent development of renal cell carcinoma (RCC) in rats and mice in high incidence and also promote renal carcinogenesis (El- Maraghy et al., 2009; Iqbal et al., 2007; Okada and Midorikawa, 1982; Mizuno et al., 2006; Jahangir and Sultana, 2006; Khan et al., 2004). Deptt. of Med. Elemn. & Toxicology 74 PhD Thesis

5 Furthermore, altered iron stores causes inflammatory conditions characterized by increased macrophage activity and enhanced neutrophil levels (Weinberg, 1992). Fe- NTA induced free radicals lead to accumulation of leukocytes in the tissue involved, and thus causes tissue injury also indirectly through activated neutrophils. Activated neutrophils are known to induce tissue injury through the production and release of reactive oxygen metabolites and cytotoxic proteins (e.g. myeloperoxidase, proteases, lactoferrin) into the extracellular fluid. Neutrophils are stimulated by various stimulants, MPO, as well as other tissue-damaging substances are released from the cells. Thus, it is an index of neutrophil infiltration (Sehirli and Sener, 2010). Since the neutrophil infiltration is an important event for the acute inflammation, increase in MPO activity due to Fe-NTA may cause inflammation and damage in the organ. Further Federico et al. (2007) reported that in most of the pathological conditions characterized by oxidative insult, there is a increase of nitrite level. NO increase the renal injury (Noiri et al., 1996) probably through its reaction with the superoxide radical (O 2 ) generating the very cytotoxic peroxynitrite (Koppenol et al., 1992), thus, both Nitric oxide and MPO are studied as inflammatory markers. Several free radical scavengers and antioxidants are effective in reducing toxic metal-mediated oxidative insult and tissue injury. Caffeic acid conferred a partially significant protection in Fe-NTA induced depletion in all the investigated antioxidant enzymes and this protection can be attributed to its antioxidant and free radical scavenging activities. We also studied the effect of caffeic acid on other biochemical parameters and early markers of tumor promotion induced by Fe-NTA. Single i.p dose induced significant depletion in the renal GSH content and its metabolizing enzymes. Depletion in GSH levels, a natural cellular antioxidant, is suggestive of the insult by toxic foreign agent (Fe-NTA). GR maintains GSH reduced where as GPx utilizes it for decomposition of lipid peroxides/hydro peroxides and other ROS. Marked decrease in the level of reduced glutathione with concomitant decrease in GR, GPx, QR, and GST levels on Fe-NTA administration (Jahangir and Sultana, 2006; Khan et al., 2004). A dose dependent marked but partially significant recovery of the depleted GR, GPx, QR, GSH and GST on pretreatment of animals with caffeic acid was observed. Lipid peroxidation and the associated membrane damage are implicated in the pathophysiology of a number of diseases including renal dysfunction (Kaur et al., 2009). Deptt. of Med. Elemn. & Toxicology 75 PhD Thesis

6 Renal dysfunction is followed by the elevated levels of serum enzymes indicating cellular leakage and loss of functional integrity of renal membrane. It correlates with our results, which showed increased activities of LDH, BUN, and Creatinine in the serum of Fe-NTA treated rats. In the present study, we show that Fe-NTA treated rats exhibit evidence of mild inflammation with elevated renal MPO and NO levels and that caffeic acid partially prevented Fe-NTA induced renal inflammation. Thus, caffeic acid attenuates these deleterious effects of Fe-NTA on the renal tissue but the change is not much significant. Oxidative stress and inflammation are closely associated with tumor promotion as well (Khan et al., 2004). Ornithine decarboxylase (ODC) is a rate limiting enzyme in the biosynthesis of polyamines, spermidine, sperimine and putricines. Elevated level of ODC activity has been consistently detected in transformed cell lines (Auvinen et al., 1992) and plays a significant role in tumor promotion (Osbrine et al., 1997). ODC activity and [ 3 H] thymidine incorporation have been widely used as biochemical marker to evaluate tumor promoting potential of an agent (Jahangir and Sultana, 2006; Khan et al., 2004). In the present study both were inhibited dose dependently by caffeic acid, suggesting its anti-tumor and antiproliferative activity potential. Inhibition of ODC activity and DNA synthesis revels that caffeic acid may intercept tumor promoting and harmful functions of polyamine biosynthesis and arachidonic acid metabolism. Further kidneys of Fe-NTA treated rats showed characteristic morphological changes such as tubular brush border loss, tubular dilation and interstitial oedema, necrosis of epithelium and hyaline casts. Treatment with caffeic acid improved deteriorated renal histo-architecture. To sum up, our present work demonstrates in Fe-NTA induced renal injuries and hyperproliferation, caffeic acid showed anti-oxidative, anti-inflammatory and anti-hyperproliferative properties to reduce lipid peroxidation, ROS generation, neutrophil infiltration and tumor promotion. We therefore postulate that the effect of ameliorating oxidative stress, inflammation and tumor promotion might be the possible mechanism by which caffeic acid acts on Fe-NTA induced renal injury. Further detailed mechanistic studies are necessary to divulge the beneficial role of caffeic acid as a useful natural, anti-cancerous antioxidant. Deptt. of Med. Elemn. & Toxicology 76 PhD Thesis

7 Table.1 Results of pretreatment of caffeic acid on antioxidant enzymes like GSH, GST, GR and GPX on Fe- NTA induced renal redox imbalance. Treatment regimen per group Group I (control) Group II (only Fe-NTA) Group III (Fe-NTA +C A D1) Group IV (Fe-NTA+ CA D2) Group V (only CA D2) GSH (n mol GSH /g tissue) GST (nmolcdnb conjugate formed/min/mg protein) GR (nmol NADPH Oxidized/min/ mg protein) GPX (n mol NA DPH Oxidized/min/ mg protein) 0.36± ± ± ± ±0.01*** 73.65±1.52** 115.5±4.27*** 156.0±2.80*** 0.33±0.02 ### 89.95±1.56 ## 145.8±1.92 # 199.8±4.80 ### 0.34±0.01 ### 89.32±4.6 ## 157.7±7.90 ## ±7.60 ### 0.39± ± ± ±8.80 Results represent mean ± SE of six animals per group. Results obtained are significantly different from Control group ( *** P < 0.001). Results obtained are significantly different from Fe-NTA treated group ( # P < 0.05), ( ## P < 0.01) and ( ### P<0001). Caffeic acid; D1= 20mg/kg/b wt; D2 = 40mg/kg/b wt. Table.2 Results of pretreatment of caffeic acid on the level of QR, catalase and SOD on Fe- NTA administration in kidney of Wistar Rats. QR γ-ggt Catalase Treatment regimen (nmolnadph (nmolespnitroaniline (nmolh per group oxidized/min/mg protein) 2 O 2 consumed formed/min/mg /min/mg protein) protein) Group I (control) ± ± ± Group II (only Fe-NTA) ± 11.96*** ± 1.30*** 803.0± 38.05*** Group III (Fe-NTA +CA D1) ± 5.85 # ± 1.86 ## ± ## Group IV (Fe-NTA+CA D2) ± ### ± 2.40 ### ± ### Group V (only CA D2) ± ± ± 7.89 Results represent mean ± SE of six animals per group. Results obtained are significantly different from Control group ( *** P < 0.001). Results obtained are significantly different from Fe-NTA treated group ( # P < 0.05), ( ## P < 0.01) and ( ### P<0001). CA, Caffeic acid; D1= 20mg/kg/b wt; D2 = 40mg/kg/b wt. Deptt. of Med. Elemn. & Toxicology 77 PhD Thesis

8 Table.3 Results of pretreatment of caffeic acid on serum markers like BUN, creatinine, LDH and SOD on Fe-NTA induced enhancement. Treatment Regimen Per Group BUN (mg/100 ml) Creatinine (mg/100ml) LDH (nmolnadhoxidized / min/mg protein) SOD (um epinehrine oxidized/min/ mg protein) GroupI (control) 35.39± ± ± ±2.91 Group II (only Fe-NTA) Group III (Fe-NTA+CAD1) Group IV (Fe-NTA+CA D2) Group V(only CA D2) 46.00±1.25*** 2.871±0.28*** ±9.18*** 113.2±2.88*** 41.39±1.01 # 2.067±0.15 # 288.9±9.6 ns 128.3±4.13 # 39.89±0.40 ## 1.979±0.12 ## 272.9±11.76 ## 132.4±4.32 ## 35.58± ± ± ±1.93 Results represent mean ± SE of six animals per group. Results obtained are significantly different from Control group ( *** P < 0.001). Results obtained are significantly different from Fe-NTA treated group ( # P < 0.05), ( ## P < 0.01) and ( ### P<0001).CA= Caffeic acid; D1= 20mg/kg/b wt; D2 = 40mg/kg/b wt. Table.4 Results of pretreatment of caffeic acid on parameters like XO, MDA and H 2 O 2 on Fe- NTA induced enhancement. TREATMENT REGIMEN PER GROUP XO (µg URIC ACID / MIN/MG PROTEIN) MDA (NMOLES OF MDA FORMED/G TISSUE) H 2 O 2 (NMOLES H202/G TISSUE) Group I (control) 0.403± ± ±11.49 Group II (only Fe-NTA) Group III (Fe-NTA +CA D1) Group IV (Fe-NTA+ + CA D2) Group V (only CA D2) 0.668±0.03*** 2.272±0.24*** 611.1±17.59*** 0.549±0.037 # 1.388±0.14 ## 545.6±13.66 ns 0.463±0.018 ## 1.154±0.08 ### 516.7±26.29 ## 0.400± ± ±10.83 Results represent mean ± SE of six animals per group. Results obtained are significantly different from Control group ( *** P < 0.001). Results obtained are significantly different from Fe-NTA treated group ( # P < 0.05), ( ## P < 0.01) and ( ### P<0001).CA= Caffeic acid; D1= 20mg/kg/b wt; D2 = 40mg/kg/b wt. Deptt. of Med. Elemn. & Toxicology 78 PhD Thesis

9 Table.5 Results of pretreatment of caffeic acid on Fe-NTA induced enhancement in hyperproliferation markers like ODC and Thymidine incorporation and inflammatory markers Like MPO and Nitrite level. Treatment regimen per group ODC (ODC activity pmol14co2relased/min/mg protein) Thymidine (Thymidine incorporation dpm/µg DNA) MPO(units of MPO activity/min/mg protein) NO (mmoles of nitrite/mg of tissue) Group I (control) 653.1± ± ± ±1.76 Group II (only Fe-NTA) Group III (Fe-NTA +CA D1) Group IV (Fe-NTA+ CA D2) Group V (only CA D2) 4771±35.22*** 4180±173.4*** 0.266±0.0** 47.80±2.43*** 1212±61.73 ## 2805±262.6 ## 0.210±0.02 # 34.60±2.18 # 886.8±20.92 ### 2340±136.6 ### 0.191±0.01 ### 26.40±2.75 ## 615.7± ± ± ±1.28 Results represent mean ± SE of six animals per group. Results obtained are significantly different from Control group ( *** P < 0.001). Results obtained are significantly different from Fe-NTA treated group ( # P < 0.05), ( ## P < 0.01) and ( ### P<0001).CA= Caffeic acid; D1= 20mg/kg/b wt; D2 = 40mg/kg/b wt. Deptt. of Med. Elemn. & Toxicology 79 PhD Thesis

Figure 1: Hematoxylin and eosin stained sections of rat kidney: (A) Normal kidney section. (B) Kidney section of Fe-NTA-treated rat showing tubular damage and brush border loss.

10 Figure 1: Hematoxylin and eosin stained sections of rat kidney: (A) Normal kidney section. (B) Kidney section of Fe-NTA-treated rat showing tubular damage and brush border loss. (C) Kidney section of caffeic acid dose 1 + Fe-NTAtreated rat showing some improvement in renal morphology (D) Kidney section of caffeic acid dose 2 + Fe-NTA-treated rat showing a near normal morphology. (40x magnification) Deptt. of Med. Elemn. & Toxicology 80 PhD Thesis

8.1. Introduction. Deptt. of Med. Elemn. & Toxicology 154 PhD Thesis

8.1. Introduction. Deptt. of Med. Elemn. & Toxicology 154 PhD Thesis 8.1. Introduction Repeated exposure to various xenobiotics like food additives, pesticides, industrial chemicals and consumer products critically effect human health causing inflammation, tissue damages