Effects of N -Acetylcysteine,Quercetin,and Phytic Acid on Spontaneous Hepatic and Renal Lesions in LEC Rats

Abstract

The effects of anti-oxidants were examined in Long-Evans Cinnamon (LEC) rats, which develop acute hepatic injury, and subsequent hepatic and renal tumors due to accumulation of excess Cu. The rats, at the age of 15 weeks, were supplied a diet containing either 1% of N-acetylcysteine (NAC), quercetin (QC), or phytic acid (PA), or basal diet alone. At weeks 2 and 6 posttreatment, animals were sacrificed for collection of blood and tissue samples. In the NAC-treated group, the development of hepatic and renal lesions was dramatically reduced. In addition, accumulation of Cu and Fe in the liver was suppressed. Acrolein-modified protein, a new marker for lipid peroxidation, was not detected in the liver or kidney of NAC treated rats, even though deposition was evident in control. Neither QC nor PA affected the development of spontaneous hepatic lesions. These results indicate that oxidative stress was reduced by NAC in the liver and kidney, and suggest that Cu and Fe may be involved in the generation of oxidative stress in the liver. In addition, it was suggested that the different effects of the anti-oxidants on lesion development in LEC rats might be related to different mechanisms of action with regard to oxidative stress.

Introduction

Long–Evans Cinnamon (LEC) rats, an inbred mutant strain, accumulate excess copper (Cu) in the liver (Sawaki et al., 1990), due to a deletion in the Cu-transporting ATPase gene (Atp7b), which is homologous to the gene responsible for human Wilson’s disease (Wu et al., 1994). They spontaneously develop acute hepatic damage, with severe jaundice, from 3–4 months after birth and approximately 20–50% die of liver injury (Yoshida et al., 1987). Almost all animals which recover develop hepatocellular carcinomas (Sawaki et al., 1990; Masuda et al., 1988) and, to a lesser degree, renal adenocarcinomas (Izumi et al., 1994). The accumulation of excess Cu is thought to be a main causal factor for acute hepatitis and hepatocellular carcinoma (Li et al., 1991), as well as renal adenocarcinomas (Kitaura et al., 1999), since treatment with Cu-chelating agents such as D-penicillamine (Togashi et al., 1992; Yikoi et al., 1994; Kitaura et al., 1999) and trientine dihydrochloride (Sone et al., 1996) effectively inhibits their development. In addition, it has been demonstrated that excess hepatic iron (Fe) accumulation also plays an important role in the liver damage in LEC rats (Kato et al., 1993, 1996).

Regarding the mechanism how Cu and Fe interact with the development of these lesions, it has been indicated that both Cu and Fe could facilitate generation of reactive oxygen species (ROS) (Aust et al., 1985; Beckman et al., 1988; Sakurai et al., 1994) and enhance hepatic lipid peroxidation (Beckman et al., 1988; Yamada et al., 1992; Yamamoto et al., 1999), resulting in oxidative hepatic damage (Sokol et al., 1990; Halliwell et al., 1992). In fact, it has been demonstrated that a spin-trapping agent, α-phenyl-tert-butylnitrone, protects against hepatic injury (Yamashita et al., 1996). In addition, an anti-oxidant, ascorbic acid, has been reported to delay the onset of jaundice (Hawkins et al., 1995). Moreover, the amount of 8-hydroxydeoxyguanosine (8-OHdG) in DNA, widely used as a marker for oxidative DNA damage, is increased in the liver and kidney of LEC rats (Yamamoto et al., 1993). Therefore, it is possible that ROS generated by excess Cu and/or Fe accumulation could be responsible for acute hepatic injury and subsequent hepatocellular carcinoma or renal adenocarcinoma development in LEC rats.

Antioxidants are widely accepted to be cancer chemopreventive agents, which have the ability to suppress production of ROS. Although N-acetylcysteine (NAC) itself is not an anti-oxidant, it provides precursor thiols for synthesis of glutathione (GSH), which plays a central physiological role in protecting cells against ROS (De Flora et al., 1995). NAC can also form stable water soluble complexes with metals (Lorber et al., 1973). Quercetin (QC), a natural plant-derived flavonoid, has been reported to scavenge ROS (Morel et al., 1993), but these 2 anti-oxidants have also been reported to cause oxidative DNA damage in vitro in the presence of Cu (Sahu and Washington, 1991; Rahman et al., 1992; Oikawa et al., 1999). Phytic acid (PA), present in plants, functions as a metal chelator in vitro (Persson et al., 1998) and is also considered to inhibit the generation of ROS. PA, in contrast with NAC and QC, has been demonstrated to effectively protect against oxidative DNA damage, even with Cu treatment in vitro (Midorikawa et al., 2001).

In the present experiment, the effects of the 3 dietary anti-oxidants on the development of spontaneous hepatic and renal lesions in LEC rats were therefore examined using serum biochemical and histopathological approaches.

Materials and Methods

Chemicals

NAC, QC, and PA were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Sigma Chemical Co. (St. Louis, USA) and Tsuno Rice Fine Chemicals (Wakayama, Japan), respectively. Their chemical structures are shown in Figure 1.

Animals and Diets

Forty male LEC rats, at 14 weeks of age, were obtained from Charles River Japan Inc. (Hino, Japan), and acclimatized for approximately 1 week before being assigned to 4 groups. They were housed in plastic cages (5 rats/cage) with soft chips for bedding in a room with a barrier system and maintained under the following conditions: temperature (23 ± 2°C), relative humidity (60 ± 5%), ventilation frequency (18 times per hour) and a 12-hour illumination per day. Normal powder diet (Oriental MF, Oriental Yeast Co., Tokyo, Japan) and tap water were available ad libitum. The Animal Care and Utilization Committee for the National Institute of Health Sciences, Japan, approved the protocols for this study.

Experimental Design

The rats were randomly divided to give equal weight distributions into 4 groups, each consisting of 10 animals. At the age of 15 weeks, they were given powdered diet containing NAC, QC or PA at a concentration of 1%, or basal diet alone. All animals were observed for clinical signs daily and body weights and food consumption were measured once every week. At weeks 2 and 6 of anti-oxidant administration, 5 animals in each group were sacrificed for collecting tissue samples and blood from the abdominal aorta under ether anesthesia.

Serum biochemical parameters, measured at SRL Inc. (Tachikawa, Japan) using sera frozen after centrifugation (set at 3,000 rpm for 10 minutes) of the collected blood, were; total protein (TP), albumin/globulin ratio (A/G), albumin (AlB), total cholesterol (T.Cho), blood urea nitrogen (BUN), cre-atinine (CRN), calcium (Ca), inorganic phosphate (P), iron (Fe), total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), copper (Cu), aspartate aminotrans-ferase (AST), alanine aminotransferase (ALT), lactate dehy-drogenase (LDH), alkaline phosphatase (AlP), γ-glutamyl transpeptidase (γ-GTP), sodium (Na), chlorine (Cl), and potassium (K).

At necropsy, all the organs were carefully examined, and the liver and kidneys were weighed, fixed, and preserved in acetone thereafter. Organs of animals found dead or sacrificed on becoming moribund were also fixed and preserved. These organs were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E), and then routine histopatho-logical examination was conducted for all animals.

Copper and Iron Staining, and Immunohistochemistry for Acrolein-Modified Proteins

For detecting copper and iron deposition, sections were stained by modified Timm’s method (Fujii et al., 1993) and Perl’s Prussian blue, respectively. Immunohistochemistry for acrolein-modified proteins was performed using an anti-acrolein-modified keyhole limpet hemocyanin monoclonal antibody, mAb5F6, at 1:10000 dilution (Uchida et al., 1998). Finally, binding was visualized using a Strept AB Complex/HRP kit (Dako Japan, Tokyo).

Statistical Analysis

Data for body weights, organ weights, and serum biochemical parameters were examined using analysis of variance (ANOVA). When positive results were obtained, the Dun-nett’s multiple comparison tests were applied to evaluate the statistical significance between pairs of groups. Data from histopathological examination and special staining were assessed by the Mann–Whitney U-test.

Results

During the experimental period, 1 animal each in the QC, PA, and control groups died or became moribund at week 6. Gross pathological findings revealed that all of these animals had severe or moderate jaundice (data not shown). Therefore, it seems likely that the cause of death was sudden hepatic injury, as spontaneously seen in LEC rats.

Body weight (Figure 2) and food consumption (data not shown) values in the control group showed gradual decrease from week 4 of the experiment, consistent with the onset of jaundice. A similar tendency was also observed in the groups given QC and PA. However, the value of body weight for the animals given NAC rather increased during the experimental period and the final body weights in this group after 6 weeks were significantly elevated (p < 0.01) as compared to the control values (Table 1). In addition, the relative liver and kidney weights of the NAC-treated group tended to be and were significantly (p < 0.01) lower than those of the control group, respectively (Table 1).

The data for serum biochemistry are summarized in Table 2. Significant differences as compared with the control group were consistently observed in the group given NAC as follows: both Fe and Cu levels were significantly decreased (p < 0.05) at week 6 as compared to the control values. In contrast with Fe, significant higher value for UIBC (p < 0.05) was observed at week 6, while UIBC exhibited a significant decrease (p < 0.05) at week 2. Regarding enzymes related to hepatic damage, a significantly lowered value for AST (p < 0.05) was noted with the NAC treatment at week 6. In the NAC-treated group, significantly elevated value for AlB (p < 0.05) was detected at week 6. As for parameters related to kidney damage, no treatment-related significant changes were noted in any of the groups.

Data for histopathological lesions that typically develop in LEC rats are summarized in Table 3. Grades of all the lesions were remarkably suppressed in the group given NAC. In the liver (Figure 3), the occurrence of enlarged hepato-cytes with large nuclei was significantly suppressed at week 2 (p < 0.05) and its severity was significantly diminished at week 6 (p < 0.01). In addition, increase in pigment granules in Kupffer cells, extramedullary hematopoiesis, oval cell proliferation, and necrosis in hepatocytes were totally suppressed, with statistical significance (p < 0.05 or 0.01), at week 6. In the kidney (Figure 4), the occurrence of tubular necrosis and hypertrophy of renal tubules was also significantly (p < 0.05 or 0.01) reduced, along with grade of eosinophilic body development (p < 0.05), at week 6. On the other hand, the severity of tubular necrosis in the group given QC was significantly enhanced (p < 0.05) at week 6 as compared with the control group.

Results of Cu and Fe staining, and immunohistochemistry for acrolein-modified proteins are summarized in Table 4. In the liver (Figure 3), both Cu and Fe were not detectable in the group given NAC, although slight accumulation of Fe was evident in the groups given QC, PA and basal diet alone (control) by week 2. Both Cu and Fe accumulation were enhanced in the groups given QC, PA or basal diet alone at week 6 as compared to the values at week 2, but still could not be detected in the group given NAC (p < 0.01) at week 6. Acrolein-modified proteins were not detected in any of the groups at week 2. At week 6, acrolein-modified protein accumulation was similarly observed in the groups given QC, PA or basal diet alone, but only at a much lower level with NAC (p < 0.01). In the kidney (Figure 4), both Cu and Fe were not detectable in any of the groups at week 2, and still could not be detected in the groups given NAC at week 6. On the other hand, Cu and/or Fe were slightly enhanced in animals receiving QC, PA, or the basal diet at week 6, though without statistical significance. Acrolein-modified proteins were not detected at week 2, but at week 6 were observed in 5/5, 2/5, 4/5, and 0/5 animals in the groups given QC, PA, basal diet alone, and NAC, respectively, the lack with the NAC group being statistically significant (p < 0.05) as compared with the basal diet group.

Discussion

The present study provided clear evidence that NAC can efficiently prevent both hepatic and renal damage in LEC rats, as assessed in terms of serum biochemistry for AST and AlB, both of which are sensitive markers for hepatic damage. Histopathological examination revealed typical lesions in the liver and kidney of LEC rats to be dramatically suppressed by NAC and free Cu and Fe concentration in the serum, which is closely associated with hepatic damage (Kato et al., 1996; Koizumi et al., 1998), was significantly reduced in contrast with the value for UIBC, by the NAC treatment. Special staining revealed accumulation of both Cu and Fe to be completely suppressed especially in the liver by NAC. It has been reported that both Cu and Fe plays an essential role in hepatic damage (Li et al., 1991; Kato et al., 1993, 1996) and both metal ions are known to be important for the production of free radicals (Aust et al., 1985; Beckman et al., 1988; Sawaki et al., 1990). From our results, we conclude that Cu and Fe might be synergistically involved in hepatic, but not renal damage.

Acrolein is now well established as a lipid peroxidation end product, produced by free radical chain reactions from fatty acids (Esterbauer et al., 1991). The aldehyde shows strong reactivity with some nucleophilic groups of amino acids and forms protein adducts (Esterbauer et al., 1991), so that acrolein-modified proteins are now accepted as markers for oxidative stress in vivo associated with lipid peroxidation. In addition, aldehydic lipid peroxidation products such as acrolein are known to yield unique DNA adducts (Chung et al., 1984). In the present study, immunohistochemistry revealed no acrolein-modified proteins in the liver or kidney of rats receiving NAC.

Regarding protective effect of NAC, it can form stable water-soluble complexes with heavy metals (Lorber et al., 1973) as well as acting as a free radical scavenger, a precursor of GSH that is readily deacetylated in cells to yield L-cysteine, thereby promoting intracellular GSH synthesis (De Flora et al., 1995). Our previous report shows that oltipraz, an an-tischistosomal agent, increased the levels of GSH in the liver of LEC rats, but spontaneous hepatic damage was nevertheless enhanced (Nakamura et al., 2002), supporting the former protecting mechanism of NAC. Therefore, the effects on both copper and iron accumulation, rather than ROS scavenging, in the target organs may explain the protective effects of NAC against oxidative stress seen in the present experiment and possibly against subsequent carcinogenesis.

In the QC treatment group, in contrast, renal tubular necrosis was slightly enhanced, although serum biochemical parameters related to kidney damage showed no significant change. Focusing on renal carcinogenicity of QC, the National Toxicology Program in the United States demonstrated the incidence of renal adenomas and adenocarcinomas to be increased in male F344 rats fed QC at the concentrations of 1% or 4% for 2 years (NTP, 1992). Together with the finding that QC can promote oxidative DNA damage in the presence of Cu in vitro (Sahu and Washington, 1991; Rahman et al., 1992), the data imply that QC might have the possibility to induce renal damage in LEC rats, even if only very weakly under the present experimental conditions.

It has been indicated that so-called “anti-oxidants” can also act as pro-oxidants in some circumstances (Inoue et al., 1992; Halliwell et al., 1995; Yamashita et al., 1998; Halliwell, 1999). With regard to 8-OHdG formation, for instance, it has been reported that NAC can cause both inhibition (Izzotti et al., 1998) and promotion (Oikawa et al., 1999) in the presence of H₂O₂ and Cu. QC also exerts promotion effects in the same conditions (Sahu and Washington, 1991; Rahman et al., 1992) while QC exerts inhibition effects in the presence of H₂O₂ only (Musonda and Chipman, 1998). Low-dose NAC may protect rats against endotoxin-mediated oxidative stress by scavenging H₂O₂, while higher doses have been found to increase mortality, so that effects are equivocal (Sprong et al., 1998). Moreover, oltipraz increased the levels of GSH in the liver of LEC rats, but spontaneous hepatic damage was nevertheless enhanced (Nakamura et al., 2002), pointing to a complex scenario in which possible anti-oxidant or pro-oxidant outcomes must be taken into account.

In the PA treatment group, we could not find any significant change related to liver damage. PA is known to be a metal chelator (Persson et al., 1998), but accumulation of both Cu and Fe was detected in the liver to the same degree as in the controls in the present study. Therefore, one possibility is that PA at the applied dose did not chelate Fe and Cu effectively in the liver under the present experimental condition. On the other hand, kidney damage was somewhat protected, accompanied by the suppression of Fe and acrolein-modified protein levels, albeit without statistical significance. Other anti-oxidants, curcumin (Frank et al., 2003), and lycopene (Watanabe et al., 2001), were earlier shown to have no preventive effects on both liver and kidney and liver carcinogenesis, respectively, in LEC rats. In addition, PA was reported to have little effect in 2-stage liver and kidney carcinogenesis models (Hirose et al., 1991; Takaba et al., 1997). However, another report demonstrated that dietary administration of PA clearly prevents diethylnitrosamine-induced rat hepatocarcinogenesis (Lee et al., 2005). Further experiments might elucidate chemopreventive effect of PA especially on the kidney.

In conclusion, NAC here caused clear reduction of both hepatic and renal damage in LEC rats, possibly by means of suppression of both Cu and Fe accumulation, especially in the liver, and oxidative stress. On the other hand, QC and PA showed very weak enhancement and reduction of renal damage, respectively, suggesting different mechanisms of action of these anti-oxidants in organs of LEC rats demonstrating spontaneous lesion development. Further studies of anti-oxidants, especially examples like NAC, regarding modification of hepatic and renal carcinogenesis in LEC rats are clearly warranted.