Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium

Toxicology 179 (2002) 37 – 50 www.elsevier.com/locate/toxicol

Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium Elisabetta Casalino, Giovanna Calzaretti, Cesare Sblano, Clemente Landriscina * Department of Pharmaco-Biology, Laboratory of Veterinary Biochemistry, Uni6ersity of Bari, Str. Pro6. per Casamassima, Km 3, 70010 Valenzano, Bari, Italy Received 22 January 2002; received in revised form 8 May 2002; accepted 27 May 2002

Abstract Catalase, Mn-superoxide dismutase (MnSOD) and Cu,Zn-superoxide dismutase (CuZnSOD) activities were studied in rat liver and kidney 6–48 h after CdCl2 intraperitoneal administration or 10 – 30 days daily oral CdCl2 intake in drinking water. This approach provided some indications as to the sensitivity of each enzyme to cadmium toxicity. These experiments showed that the formation of thiobarbituric acid reactive substance (TBARS) did not strictly depend on how well the antioxidant enzyme worked. From in vitro experiments it appeared that TBARS removal by vitamin E did not restore the three enzyme activities at all. As for cadmium’s inhibitory mechanism on catalase activity, our data, obtained in the pH range 6.0–8.0, are a preliminary indication that the negative effect of this metal is probably due to imidazole residue binding of His-74 which is essential in the decomposition of hydrogen peroxide. Cadmium inhibition of liver mitochondrial MnSOD activity was completely removed by Mn2 + ions, suggesting that the reducing effect on this enzyme is probably due to the substitution of cadmium for manganese. We also observed the antioxidant capacity of Mn2 + ions, since they were able to normalize the increased TBARS levels occurring when liver mitochondria were exposed to cadmium. The reduced activity of CuZnSOD does not seem to be due to the replacement of Zn by Cd, nor to the peroxides formed. As this enzyme activity was almost completely recovered after 48 h, we hypothesize that the momentary inhibition is imputable to a cadmium/enzyme interaction. This causes some perturbation in the enzyme topography which is critical for its catalytic activity. The pathological implications linked to antioxidant enzyme disorders induced by cadmium toxicity are discussed. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cadmium in vivo toxicity; Manganese as preventing agent; Antioxidant enzymes; Cadmium in vitro mechanism

1. Introduction * Corresponding author. Tel.: + 39-080-5443864; fax: + 39080-5443863 E-mail address: [email protected] (C. Landriscina).

Cadmium, a potent toxic metal, is very harmful to the environment and to humans because of in vivo accumulation in liver, kidney and other tissues. The toxicity of cadmium as an industrial

0300-483X/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 2 4 5 - 7

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E. Casalino et al. / Toxicology 179 (2002) 37–50

pollutant, a food contaminant and as one of the major components in cigarette smoke has been well established (Morselt, 1991). Cadmium accumulates and proves to be very toxic in many organs, such as kidney, liver, lung, testis, brain, bone, blood system, etc. (WHO, 1992; U.S. Department of Health and Human Services, 1997). The adverse effect of this metal has also been linked to neoplastic diseases and aging (Goyer, 1989; Saygi et al., 1991). The molecular mechanism responsible for the toxic effects of cadmium is far from being completely understood. Suggestions of the type of damage involved in Cd-induced cellular toxicity include: interference with antioxidant enzymes (Hussain et al., 1987; Shukla et al., 1987), alterations in thiol proteins (Chan and Cherian, 1992; Li et al., 1993), inhibition of energy metabolism (Muller, 1986), alteration in DNA structure (Coogan et al., 1992), altered membrane structure/function (Muller, 1986; Shukla et al., 1987). Cadmium induces the expression of a number of stress genes (Wang and Templeton, 1998; Goering et al., 1993), and plays an important role in affecting some enzymatic activities (Jay et al., 1991; Watjen et al., 2001; Casalino et al., 2000; Manca et al., 1991; Casalino et al., 1997). Since it causes lipid peroxidation in numerous tissues both in vivo and in vitro (Muller, 1986; Ognjanovic et al., 1995; Casalino et al., 2000), it has been suggested that cadmium’s action is indirect and that the metal most probably responsible for this process is the iron removed from membranes (Casalino et al., 1997). Lipid peroxidation has long been considered the primary mechanism for cadmium toxicity (Muller, 1986; Manca et al., 1991; Yiin et al., 1999), despite its inability to directly generate free radicals under physiological conditions (Eneman et al., 2000). The intracellular concentration of reactive radical species can be increased either by overproduction of reactive oxygen species (ROS) or by an inability of the metabolic system to destroy them. Conflicting results have been reported on the activities of antioxidant enzymes in oxidative stress induced by cadmium in various organs (Shukla et al., 1989; Bjerrum et al., 1991; Sarkar et al., 1995; Stajn et al., 1997). Results both from our laboratories and from others have

indicated that cadmium toxicity induced enzyme dysfunction is imputable to mechanisms that are unlike lipid peroxidation, such as metal enzyme substitution or cadmium/apoenzyme interaction (Casalino et al., 2000; Strubelt et al., 1996). Since the role of antioxidant enzymes in lipid peroxidation caused by cadmium appears controversial, in the present study we report results on lipid peroxidation and the activity of catalase, Mn superoxide dismutase (MnSOD) and Cu,Zn-superoxide dismutase (CuZnSOD) in rat liver and kidney at different times following intraperitoneal or oral cadmium intoxication. A number of different behavior patterns have emerged, as regards the period of time and the amount of cadmium inhibition, between liver and kidney MnSOD and catalase activities on one hand and CuZnSOD on the other. Preliminary information on cadmium’s inhibitory mechanism on antioxidant enzymes was obtained from in vitro experiments. These experiments also showed that the antioxidant capacity of vitamin E is unable to restore reduced enzyme activities and that manganese ions, as well as removing the cadmium inhibition of MnSOD, have an active antioxidant capacity.

2. Materials and methods

2.1. Reagents All chemicals used were of the highest quality and purchased from BDH Chemicals, Fisher Scientific Supply or Sigma Chemical Co. Chelex 100 ion exchange resin (Bio-Rad Laboratories) was used to remove contaminating metals from all reagents. Biochemicals were obtained from Boehringer, Mannheim. Organic solvents of analytical grade were used. All solutions were prepared in double-distilled water.

2.2. Animal treatment Single male Wistar rats weighing 180 –200 g and maintained on a standard diet were injected i.p. with a single dose of 2.5 mg Cd/kg body weight, as CdCl2, in 0.1 ml saline and killed 6, 24 and 48 h after injection. Single control animals

E. Casalino et al. / Toxicology 179 (2002) 37–50 39

received the equivalent volume of saline. In another set of experiments, rats received cadmium in drinking water (250 mg/l CdCl2, in tap water) and were killed after 10, 20 and 30 days of treatment. The control animals received only tap water. In each experiment, a pair of animals (one control and one Cd-treated rat) were used. The animals were sacrificed by decapitation under ether anesthesia. The livers and kidneys were quickly excised, rinsed in ice-cold saline to clear them of blood, weighed, finely minced in the same solution, and homogenized (approximately 10% w/v) in a Potter Elvehjem homogenizer with a Teflon pestle. Liver and kidney homogenate were used for thiobarbituric acid reactive substance (TBARS) determination; mitochondria and postmitochondrial supernatant from both control and Cd-treated rats, obtained by differential centrifugation as reported previously (Landriscina et al., 1976) were used for enzyme assays.

2.3. In 6itro experiments Liver and kidney mitochondria and postmitochondrial supernatant from untreated rats were obtained as indicated above. The incubation was performed at 37 °C for 20 min in a mixture containing 75 mM CdCl2, 0.175 M KCl, 25 mM Tris –HCl at pH 7.4 or as indicated elsewhere. Two hundred micromolar vitamin E (dissolved in 25 ml of dimethyl sulfoxide to a final volume of 1 ml) and 30 mM MnSO4, when used, was added 30 s before CdCl2. The reaction was stopped on ice. Aliquots of the suspension were used to determine lipid peroxidation and enzyme activities.

2.5. Enzyme assay Enzyme activities were measured using a temperature-controlled Beckman DU 640 spectrophotometer. The assays were run in duplicate or triplicate. Catalase (EC 1.11.1.6) activity was assayed by following the decrease of H2O2 at 240 nm (Clairborne, 1985). Superoxide dismutase (SOD, EC 1.15.1.1) activities were determined from their ability to inhibit the autoxidation of epinephrine. Stimulation of epinephrine autoxidation by traces of heavy metals present as contaminants in the reagents or by the other metals under investigation was prevented by adding 10 − 4 M EDTA in the buffer to chelate these ions (Misra, 1985). The two isoforms of SOD were distinguished by cyanide activity inactivation, CuZnSOD being strongly inactivated unlike MnSOD whose activity was slightly reduced by cyanide. One unit of CuZnSOD and MnSOD is defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50%.

2.6. Protein concentration The protein content in mitochondria and postmitochondrial supernatant was estimated by the biuret method (Gornall et al., 1949) using bovine serum albumin as standard.

2.7. Statistics Statistical evaluation of the data was performed using the Student’s t-test. Differences from controls were considered significant at PB 0.05.

2.4. Lipid peroxidation

3. Results

Lipid peroxidation was measured by determining the TBARS concentration in the presence of BHT (butylated hydroxytoluene) (0.03%, final concentration) under continuous nitrogen flow to avoid any artifactual oxidation due to heating (Buege and Aust, 1987). In the in vivo experiments, TBARS concentrations were measured in homogenate in the same way.

In cadmium-intoxicated rats, hepatic and renal TBARS production shows similar increases at short time intervals (6 h) after i.p. metal administration (Table 1), when 30% more is found in both organs than in the control. After 24 h this increase is much higher both in liver (82%) and kidney (67%). A difference can be observed after longer time intervals (48 h) when the TBARS level

37.793.9 (4)*

33.793.5 (4)*

24

48

50.79 5.2 (4)****

59.89 5.7 (4) 54.49 5.3 (4)**** 44.3 9 4.1 (4)**

CuZnSODb

26.1 92.5 (4)*

25.7 9 2.9 (4)*

383.0 9 39.1 (4) 49.6 9 5.2 (4) 364.7 937.1 64.9 9 6.3 (4)** (4)**** 243.1 923.8 82.8 98.7 (4)* (4)* 204.1 921.2 51.1 9 4.9 (4)* (4)****

44.5 9 4.5 (4) 27.8 92.6 (4)*

TBARSa

Catalaseb

Mn SODb

Kidney

36.7 93.5 (4)****

42.8 9 4.1 (4) 37.8 93.9 (4)**** 22.5 92.3 (4)*

CuZnSODb

27.4 93.0 (4)***

16.4 9 1.8 (4)*

34.2 93.3 (4) 22.59 2.4 (4)**

MnSODb

98.6910.3 (4)*

158.2 9 16.1 (4) 149.1 915.2 (4)**** 89.4 9 9.1 (4)*

Catalaseb

Data represent the mean of eight experimental values 9SD. The number in parentheses indicate the number of experiments in duplicate. In each experiment single rat was injected i.p. with CdCl2 dissolved in 1.0 ml physiological solution (2.5 mg CdCl2/kg body weight) and sacrificed after the indicated times. Control rat received the same volume of saline. CuZnSOD and catalase activities were measured in postmitochondrial supernatant while MnSOD activity was measured in mitochondria from control and Cd-treated rats. a Expressed as nmol/g tissue. b Specific activity expressed as U/mg protein. * PB0.001. ** PB0.01. *** PB0.05. **** P= N.S.

20.79 2.2 (4) 28.6 92.9 (4)**

Control 6

Time after CdCl2 Liver administration (h) TBARSa

Table 1 TBARS level and antioxidant enzyme activities in rat liver and kidney at different times after cadmium administration

40 E. Casalino et al. / Toxicology 179 (2002) 37–50

23.89 2.5 (4) 27.49 2.6 (4)***** 28.8 93.1 (4)**** 25.19 2.6 (4)***** 48.094.5 (4)*****

39.49 4.1 (4)**

53.49 6.1 (4) 37.99 3.9 (4)**

25.9 9 2.4 (4)**

27.2 92.9 (4)**

408.2 9 39.1 (4) 54.8 95.6 (4) 341.7 935.5 60.3 96.2 (4)**** (4)***** 314.5 931.8 65.5 96.1 (4)** (4)**** 318.4 930.1 58.6 96.2 (4)*** (4)*****

38.2 93.6 (4) 25.5 92.7 (4)**

TBARSb

Catalaseb

MnSODb

TBARSa

CuZnSODb

Kidney

Liver

32.2 9 3.4 (4)*****

24.6 9 2.7 (4)**

37.8 9 4.0 (4) 19.7 9 2.1 (4)*

CuZnSODb

17.2 9 1.8 (4)*

21.89 2.4 (4)**

28.9 92.7 (4) 21.19 2.3 (4)**

MnSODb

181.2 9 19.2 (4) 121.5911.8 (4)** 126.19 13.1 (4)** 136.1 9 12.6 (4)**

Catalaseb

Data represent the mean of eight experimental values 9 SD. The number in parentheses indicate the number of experiments in duplicate. CdCl2 was administered to rats in drinking water (250 mg/l) for the indicated times. Control rats received only tap water. Enzyme activities were measured in the respective subcellular fractions as indicated in Table 1. a Expressed as nmol/g tissue. b Specific activity expressed as U/mg protein. * PB0.001. ** PB0.01. *** PB0.02. **** PB0.05. ***** P= N.S.

30

20

Control 10

Time after CdCl2 administration (days)

Table 2 Liver and kidney TBARS level, catalase and superoxide dismutase activities in rats intoxicated with cadmium given in the drinking water

E. Casalino et al. / Toxicology 179 (2002) 37–50 41

E. Casalino et al. / Toxicology 179 (2002) 37–50

42

still remains very high in liver (more than 63%), while in kidney it falls to a level similar to that in the control. As for the activities of the three antioxidant enzymes, CuZnSOD, catalase and MnSOD, they exhibit similar behaviour patterns in both organs at all times after cadmium intoxication. Indeed, in Table 1 it is shown that the activity of both hepatic and kidney CuZnSOD is almost unaffected after both short (6 h) and long intervals (48 h), while after 24 h it is reduced by 26 and 48%, respectively. Catalase activity, too, is not significantly influenced 6 h after cadmium administration; however, after longer intervals (24 and 48 h), it remains strongly inhibited in both organs. Mitochondrial MnSOD, unlike catalase and CuZnSOD, is strongly reduced at all times following cadmium intoxication both in liver and in kidney, where an inhibition ranging from 38 to 52% is found, with the exception of a lower value (20%) in kidney after 48 h. When TBARS production and the activities of the three antioxidant enzymes are measured in the liver and kidney of cadmium-intoxicated rats given CdCl2 in drinking water for between 10 and 30 days, a different situation is observed. The

data in Table 2 indicate that, after 10 days of cadmium administration, liver and kidney TBARS levels are not significantly elevated, while the activities of CuZnSOD, catalase and MnSOD are reduced in both organs. After 20 days of intoxication, the three hepatic and renal enzyme activities remain inhibited by about the same amount, while TBARS production in both organs is only 21% higher than in untreated rats. After increasing the time to 30 days, liver and kidney CuZnSOD activity and TBARS levels are similar to those in the control, while both MnSOD and catalase activities remain reduced. Data in Tables 3 and 4 indicate that the activities of liver and kidney CuZnSOD, catalase and MnSOD are reduced when postmitochondrial supernatant or mitochondria are incubated with CdCl2. CuZnSOD and MnSOD activities are reduced more in liver subcellular fractions than in kidney, while the amount of catalase inhibition is the same in the postmitochondrial supernatant of both organs. The situation concerning TBARS formation in liver and kidney subcellular fractions following incubation with cadmium is, however, very different. In liver subcellular fractions,

Table 3 Rat liver catalase and superoxide dismutase activities in the presence of cadmium: the role of vitamin E Additions

Postmitochondrial supernatant

Mitochondria

TBARS (nmol/mg protein)

CuZnSOD activitya

Catalase activitya

TBARS (nmol/mg protein) MnSOD activitya

Control CdCl2

0.21 90.02 (3) 0.62 90.06 (3)*

40.2 94.3 (3) 26.7 93.0 (3)***

0.42 9 0.05 (3) 0.98 9 0.10 (3)*

34.5 93.5 (3) 20.4 92.6 (3)**

Vitamin E

0.20 9 0.02 (3)*****

39.79 4.2 (3)***** 26.2 93.1 (3)***

348.6 9 35.1 (3) 270.6 926.9 (3)**** 337.1 9 31.4 (3)***** 263.7 927.1 (3)****

0.41 9 0.04 (3)*****

35.8 93.7 (3)***** 19.3 9 2.0 (3)**

CdCl2+vitamin 0.22 9 0.03 (3)***** E

0.43 90.05 (3)*****

Data represent the mean of six experimental values 9SD. The number in parentheses indicate the number of experiments in duplicate. The activity of the above enzymes was measured in postmitochondrial supernatant and mitochondria from untreated rats. Incubation time, 20 min at 37 °C. Incubation medium, 0.175 M KCl, 25 mM Tris–HCl, pH 7.4 (final volume, 1 ml). Postmitochondrial supernatant protein, 3 mg/ml; mitochondrial protein, 3 mg/ml. a Specific activity expressed as U/mg protein. * PB0.001. ** PB0.01. *** PB0.02. **** PB0.05. ***** P =N.S.

E. Casalino et al. / Toxicology 179 (2002) 37–50

43

Table 4 Rat kidney catalase and superoxide dismutase activities in the presence of cadmium: the role of vitamin E Additions

Postmitochondrial supernatant

Mitochondria

TBARS (nmol/mg protein)

CuZnSOD activitya

Catalase activitya

TBARS (nmol/mg protein) MnSOD activitya

Control CdCl2

0.39 90.04 (3) 0.61 90.07 (3)*

38.69 3.9 (3) 27.792.6 (3)**

0.69 9 0.08 (3) 0.90 90.10 (3)***

27.4 92.5 (3) 18.8 9 1.7 (3)*

Vitamin E

0.35 90.03 (3)****

38.29 3.6 (3)**** 28.1 92.7 (3)**

139.9 912.8 (3) 107.8 911.2 (3)*** 144.5 9 14.6 (3)**** 105.5 9 9.6 (3)***

0.62 9 0.06 (3)****

28.1 9 2.6 (3)****

0.71 9 0.07 (3)****

18.1 9 1.9 (3)*

CdCl2+vitamin 0.38 90.04 (3)**** E

Data represent the mean of six experimental values 9SD. The number in parentheses indicate the number of experiments in duplicate. Experimental conditions as described in Table 3. a Specific activity expressed as U/mg protein. * PB0.01. ** PB0.02. *** PB0.05. **** P= N.S.

TBARS production is about four times higher than in kidney fractions (an increase of 195 and 133% in liver postmitochondrial supernatant and mitochondria, respectively, compared to an increase of 56 and 30% in the corresponding kidney fractions). Another interesting aspect shown in these two tables is that the TBARS level found in liver and kidney fractions following incubation with CdCl2 is practically eliminated, being equal to that of the control in the presence of vitamin E. Removal of peroxides by vitamin E does not prevent cadmium inhibition of the three enzyme activities under investigation, indicating that peroxides are not involved in the inhibitory process of these enzymes. In order to gain some insight into cadmium’s inhibitory mechanism, we investigated the effect of increasing CdCl2 concentrations on catalase activity in liver and kidney postmitochondrial supernatant in the pH range 6.0– 8.0. As for the optimal pH of catalase, from the results in Table 5 in the absence of CdCl2 it is possible to see that the enzyme works well in this pH range. Here it is shown that, at pH 6.0, the highest enzyme inhibition observed is less than 20% following 100 mM

CdCl2 incubation with both tissue fractions. By contrast, at any cadmium concentrations, tested at pH 7.4 and 8.0 on the same tissue fractions, a much higher level of enzyme inhibition is observed. In these conditions, 100 mM CdCl2 reduces enzyme activity by 31.0 and 42.1% in liver and by 28.1 and 39.1% in kidney at pH 7.4 and 8.0, respectively. This seems to indicate that between pH 7.4 and 8.0, catalase begins to interact negatively with cadmium. The effect of Mn2 + ions on rat liver mitochondria lipid peroxidation and MnSOD activity following cadmium incubation is described in Table 6. The data, in agreement with previous reports (Coassin et al., 1992; Knight and Searles, 1994), indicate that Mn2 + ions exhibit an antioxidant activity since, on incubating subcellular liver particles with cadmium in the presence of 30 mM MnSO4, a TBARS amount similar to that of the control is found. In addition, MnSOD activity, which is strongly reduced by cadmium, is promptly restored when the same MnSO4 concentration is present in the incubation medium, indicating that some competition probably occurs between Cd2 + and Mn2 + ions in enzyme binding.

355.5 935.6 (4) 346.3 935.1 (4)**** 308.0929.8 (4)**** 295.89 27.6 (4)***

16.8

13.4

2.6

–

378.4 938.1 (4) 302.7 930.5 (4)*** 282.19 27.6 (4)** 261.59 25.8 (4)** 31.0

25.5

20.0

–

Decrease (%) 360.19 36.4 (4) 266.0 927.2 (4)** 247.7 923.4 (4)** 208.7 9 19.1 (4)*

8.0

42.1

31.2

26.1

–

Decrease (%) 142.2915.1 (4) 135.3913.9 (4)**** 121.5 912.2 (4)**** 114.7 910.9 (4)*** 19.4

14.5

4.8

–

Decrease (%)

6.0

7.4

6.0

Decrease (%)

Kidney specific activitya pH

Liver specific activitya pH

146.8914.8 (4) 122.8 911.9 (4)*** 112.7 910.5 (4)** 105.5 99.2 (4) **

7.4

28.1

23.2

16.3

–

Decrease (%)

146.89 13.9 (4) 115.0 9 10.1 (4)** 96.39 8.9 (4)* 89.4 99.1 (4)*

8.0

39.1

34.4

21.7

–

Decrease (%)

Data represent the mean of eight experimental values 9 SD. The number in parentheses indicates the number of experiments in duplicate. Experimental condition as described in Table 3. Catalase activity was measured in postmitochondrial supernatant from untreated rats. a Specific activity expressed as U/mg protein. * PB0.001. ** PB0.01. *** PB0.05. **** P =N.S.

100

75

50

0

CdCl2 (mM)

Table 5 Effect of increasing cadmium concentrations on rat liver and kidney catalase activity in the pH range 6.0–8.0

44 E. Casalino et al. / Toxicology 179 (2002) 37–50

E. Casalino et al. / Toxicology 179 (2002) 37–50

4. Discussion Cadmium toxicity-induced lipid peroxidation has been extensively studied in several laboratories (Hussain et al., 1987; Manca et al., 1991; Casalino et al., 1997), but its peroxidative mechanism is a controversial matter, as cadmium does not undergo redox cycling and probably acts through indirect mechanisms (Casalino et al., 1997; Yiin et al., 2001). There is evidence that ROS are formed in the presence of cadmium, and these could be responsible for its toxic effects (Amoroso et al., 1982; Bagchi et al., 1996; Thevenod and Friedmann, 1999; Szuster-Clesielska et al., 2000). It has been proposed that the enhancement of lipid peroxidation by cadmium in rats is a consequence of a decrease in superoxide dismutase and catalase activities (Hussain et al., 1987; Shukla et al., 1989; Ognjanovic et al., 1995). However, some laboratories have reported that high levels of lipid peroxides were found in rat tissues following cadmium exposure, but that superoxide dismutase activities increased (Sarkar et al., 1995; Zikic et al., 1998) and no significant change in catalase activity occurred (Sarkar et al., 1995; Stajn et al., 1997). Our results, at different times following intraperitoneal or oral cadmium intoxication (Tables 1 and 2), indicate that an increased TBARS level sometimes corresponds to an almost regular antioxidant enzyme activity, while in other conditions a reduced antioxidant activity is followed by a normal TBARS concentration both in liver and kidney. We hypothesize

45

that the factors responsible for some peroxidative processes in liver and kidney are probably not strictly dependent on how well the antioxidant enzymes work, as also shown by other authors (Hussain et al., 1987; Strubelt et al., 1996; Manca et al., 1994; Nigam et al., 1999). The fact that in vivo a high TBARS level is formed when the antioxidant enzymes are functioning normally indicates that cadmium-induced lipid peroxidation does not strictly depend on ROS. The low or insignificant TBARS increase measured in chronic cadmium intoxication when antioxidant enzyme activity is reduced is probably due to the activation of other defence factors. Several protective agents, including glutathione and metallothionein, play an important role in detoxification processes (Hayes and McLellan, 1999; Cherian, 1994; Brugnera et al., 1994). Following chronic exposure to cadmium, an increased hepatic glutathione level has been found (Congiu et al., 2000), probably as a consequence of inducing the expression of gamma-glutamylcysteine synthetase, which catalyzes the rate-limiting reaction of the biosynthesis of this tripeptide (Griffith, 1999; Wild and Mulcahy, 2000; Shukla et al., 2000). An induced metallothionein synthesis has also been observed in various rat tissues after exposure to cadmium (Onosaka and Cherian, 1981; Brzoska et al., 2000). In vitro experiments on postmitochondrial supernatant from liver and kidney indicated that the order of cadmium’s inhibitory effect on enzyme activities is MnSOD\ CuZnSOD \ catalase and

Table 6 Antioxidant effect of Mn2+ ions and their capacity to remove cadmium MnSOD activity inhibition in rat liver mitochondria Additions

TBARS (nmol/mg protein)

%

MnSOD (U/mg protein)

%

Control CdCl2 MnSO4 CdCl2+MnSO4

0.4690.05 0.8890.09 0.44 90.05 0.45 90.05

+91.3 −4.3 −2.2

35.6 94.1(4) 21.7 9 2.5 (4)** 42.6 93.8 (4)*** 39.4 94.2 (4)****

−39.0 +19.7 +10.7

(4) (4)* (4)**** (4)****

Data represent the mean of eight experimental values 9 SD. The number in parentheses indicates the number of experiments in duplicate. Mitochondria from untreated rats have been used. Experimental condition as described in Table 3. * PB0.001. ** PB0.01. *** PB0.05. **** P= N.S.

46

E. Casalino et al. / Toxicology 179 (2002) 37–50

that TBARS removal by vitamin E does not restore the reduced enzymes at all (Tables 3 and 4). On the basis of the in vivo response of these enzymes to cadmium intoxication (Tables 1 and 2), our results seem to indicate that there is no tissue-specific effect on enzyme activity. Our data agree with previous findings indicating that the antioxidant alpha-lipoic acid decreases lipid peroxidation but does not affect cadmium-inhibited catalase activity (Bludovska et al., 1999); however, they are at variance with other findings that vitamin E, besides a significant reduction in lipid peroxides, can reverse the inhibitory effect of cadmium on antioxidant enzymes (Sarkar et al., 1997; El-Missiry and Shalaby, 2000). In our opinion the experiments described in Tables 5 and 6 represent the initial attempt to explain the molecular mechanisms by which cadmium reduces antioxidant enzymes under investigation. The increase in cadmium’s inhibitory effect on catalase activity on increasing the pH from 6.0 to 8.0 in both liver and kidney postmitochondrial supernatant (Table 5) suggests that raising pH causes some variation in enzyme conformation and that this favours the negative action of cadmium. The structure of catalase, as well as its catalytic mechanism, has been well examined in many species (Kirkman and Gaetani, 1984; Fita and Rossmann, 1985). This enzyme is a tetramer with four heme groups per tetramer. The N delta of the His-74 imidazole residues is essential for its catalytic activity. Through its participation in hydrogen bonding with other residues in the substrate pocket, the N delta facilitates the decomposition of hydrogen peroxide into water and oxygen (Fita and Rossmann, 1985; Murthy et al., 1981). The reduction of the protonation state of nitrogen in the imidazole residue of His-74 (pKa equal to 6.5) when raising the pH to physiological value (Table 5) probably makes it more feasible for Cd2 + to interact with this unprotonated nitrogen, thus reducing enzyme activity. The reduction in in vivo catalase activity is probably imputable to this interaction, since inhibition lasts for a long time after intoxication. The reduced catalase activity seen in vivo both in liver and kidney 24 h after intraperitoneal cadmium injection (Table 1) is probably due to the time

needed for an active interaction between cadmium and apoenzyme to be set up at physiological pH. The pH-dependent inhibition of catalase by cadmium is similar to previously reported results regarding NADPH– cytochrome P 450 reductase, an enzyme containing histidine in the active site, whose activity is strongly reduced only at pH 7.4 and above, and not at lower pH levels (Casalino et al., 2000). MnSOD is a homotetrameric enzyme that protects mitochondria against oxygen-mediated free radical damage. The considerable decrease in the activity of this enzyme both in liver and kidney already occurs 6 h after intraperitoneal cadmium injection and lasts for a long time (Table 2). This effect is probably due to cadmium substituting manganese, since enzyme activity was totally restored by incubating mitochondria with cadmium plus Mn2 + (Table 6). Following a long period of cadmium intoxication, MnSOD activity was found to be strongly reduced in both rat liver and kidney, and, on the basis that in vitro cadmium also strongly inhibited this enzyme, a non-specific interaction between cadmium and MnSOD was proposed (Hussain et al., 1987). Moreover, our data, indicating that mitochondria incubated with Mn2 + show an increased MnSOD activity agree, with previous results showing that MnSOD activity increases in the brain of manganese-treated adult rats (Hussain and Ali, 1999). The concomitant antioxidant effect of Mn2 + ions observed in mitochondria incubated with CdCl2 is not due to stimulated MnSOD activity, since Mn2 + ions also exerted the same antioxidant effect on rat liver microsome lipid peroxidation induced by ascorbate or NADPH (data not shown; see also Coassin et al., 1992; Knight and Searles, 1994). The antioxidant activity of Mn2 + has also been observed in isolated hepatocytes, in which cell injury by cadmium was much reduced in the presence of manganese (Stacey and Klaassen, 1981). As for manganese’s antioxidant activity, it has been proposed that in particular experimental conditions Mn2 + ’s oxidation potential can shift from 1.9 V to lower values (Kozlov et al., 1997). This is probably the case in our conditions, as electrons can be donated from manganese ions to peroxidation-by-products.

E. Casalino et al. / Toxicology 179 (2002) 37–50

When the cytosolic activity of CuZnSOD is measured in rat liver and kidney at different intervals following intraperitoneal cadmium administration, a completely different behaviour from that shown by MnSOD is observed. CuZnSOD activity is negatively affected only 24 h after intoxication and appears almost normalized after 48 h (Table 1). Likewise, after 30 days of daily oral cadmium intake, CuZnSOD activity is similar to the control. Otherwise in other laboratories it has been found that CuZnSOD activity is strongly reduced in rat liver and kidney after chronic cadmium administration (Hussain et al., 1987; Stajn et al., 1997). Anyhow, in vitro studies have indicated that CuZnSOD activity, like MnSOD activity, is strongly inhibited by cadmium (Table 3). The possibility has been advanced that cadmium can replace Zn, thus reducing superoxide dismutase activity (Hussain et al., 1987; Muller, 1986; Kofod et al., 1991; Bauer et al., 1980). In our experimental conditions, we did not obtain such evidence, since the addition of Zn2 + ions failed to restore CuZnSOD activity in both hepatic and renal postmitochondrial supernatant exposed to cadmium (data not shown). Other authors have suggested free radical-mediated inactivation of CuZnSOD due to enzyme protein fragmentation (Kwon et al., 2000). In the present study, we exclude this hypothesis since, following TBARS removal by vitamin E, CuZnSOD activity still remains reduced by cadmium both in liver and kidney postmitochondrial supernatant (Tables 3 and 4). As the Cu2 + ion is the active site of CuZnSOD, localized at the bottom of a deep channel where the superoxide anion is electrostatically driven (Tainer et al., 1983; Fisher et al., 1991), we hypothesize that the cadmium/enzyme interaction probably causes a transient perturbation to the topography of this channel which is critical to the enzyme’s function. Alternatively, cadmium may alter the protein conformation by interacting with the enzyme, thereby altering its functional activity, analogously to the impairment caused by this metal to p53, a tumor-suppressor protein involved in the control of cell cycles (Meplan et al., 1999). In summary, this study provides the first indication on the possible mechanism by which cad-

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mium inhibits MnSOD and catalase, also showing that this metal affects mitochondrial MnSOD in a different way from cytosolic CuZnSOD. As many metabolic disorders have been associated with the impairment of these enzymes, a major investigation needs to be carried out in this area. Nephrotoxicity induced by cadmium has been attributed to an excess of ROS (Marumo and Li, 1996; Thevenod and Friedmann, 1999) probably due to the reduction in superoxide dismutase activity. This oxidative damage causes the degradation of Na+/K+-ATPase, thus contributing to the Fanconi syndrome. It has recently been reported that cadmium exposure causes amyotrophic lateral sclerosis, due to reduced brain superoxide dismutase (Bar-Sela et al., 2001), and damages the basal ganglia, resulting in parkinsonism (Okuda et al., 1997). Indeed in patients with neuropsychiatric disorders (Ravikumar et al., 2000) and Parkinson’s disease (de la Torre et al., 1996), the activities of superoxide dismutase, catalase and other enzymes involved in free radical scavenging are decreased.

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