Dietary cadmium decreases lipid peroxidation in the liver and kidneys of the bank vole (Clethrionomys glareolus)

J. Trace Elements Med. Biol. 14, pp. 76-80 June 2000

Journal o

Trace Elements © 2000 Urban & Fischer

Dietary cadmium decreases lipid peroxidation in the liver and kidneys of the bank vole

( Clethrionomys glareolus) T. Wlostowski 1, A. Krasowska and B. Godlewska-Zylkiewicz* Institute of Biology, University of Bialystok, Swierkowa 20B, 15-950 Bialystok, Poland *Institute of Chemistry, University of Bialystok, Pilsudskiego 11/4, 15-950 Bialystok, Poland

Summary

It is not known, however, whether Cd affects hepatic and reuaI LPO when administered orally as dietary Cd. Therefore, the

The effect of elevated levels of dietary cadmium on lipid peroxidation in the liver and kidneys of a small rodent, the bank vole, was determined in the present study. Males and females, aged 1 month, were given diets containing 0.40 and 80 mg Cd per kg; liver and kidneys were removed for TBA-RS as well as iron, copper, zinc, cadmium and metallothionein analyses at the end of 6 weeks. Dietary Cd significantly decreased the TBA-RS level in the liver and kidneys of both sexes; however, this effect appeared to be dose-dependent only for the male liver. The changes in hepatic and renal TBA-RS paralleled closely those of tissue iron. Copper concentration decreased significantly only in the male liver, while hepatic and renal zinc were not influenced by dietary Cd. The concentrations of Cd and metallothionein in the liver and kidneys increased significantly in a dose-dependent fashion. Regression analysis confirmed that TBA-RS in both organs correlated closely with iron. The data suggest that dietary Cd decreases hepatic and renal lipid peroxidation indirectly, through lowering the tissue iron concentration.

main purpose of the present work was to examine LPO in the liver and kidneys of a small rodent, the bank vole, exposed subchronically to elevated levels of dietary Cd; the rodent is used frequently in both laboratory and field studies concerning various toxicological issues (5, 6, 7). Since the tissue iron (Fe), copper (Cu), zinc (Zn) and metallothionein (MT) are important determinants of LPO (8, 9, 10, 11, 12, 13) and their status is essentially affected by dietary Cd (14, 15), the concentrations of Fe, Cu, Zn and MT in both organs were also determined to establish what relationship, if any, existed between these elements and LPO.

Materials and Methods Animals and experimental design

Keywords: lipid peroxidation, cadmium, iron, copper, metal-

Male and female bank voles from our own laboratory stock

Iothionein Abbreviations: AAS: Atomic absorption spectrophotometry, ANOVA: Analysis of variance, DM: Dry matter, FM: Fresh matter, In: Natural logarithm, LPO: Lipid peroxidation, MDA: Malondialdehyde, MT: Metalrothionein, ROS: Reactive oxygen species, TBA-RS: Thiobarbituric acid-reactive substances. (Received October 1998/May 1999)

were used throughout the study. One-month-old voles, weighing 10-13 g, were randomly allocated into three groups (n = 8

Introduction The studies carried out so far indicate that cadmium (Cd) increases lipid peroxidation (LPO) in isolated hepatocytes (1) as well as in the liver and kidneys of Cd-injected animals (2, 3, 4). 1To whom correspondence should be addressed.

each) according to dietary Cd: (a) control, (b) Cd-40 and (c) Cd80 mg/kg DM. The animals were housed in groups of four in stainless-steel cages and kept for 6 weeks on a 12 h light/dark cycle in a room maintained at 18-20°C and at 50-70% relative humidity. For 6 weeks, the bank voles received ad libitum distilled water and clean (control) or Cd-containing wheat grains which are considered to be an adequate quality food for the bank vole (16). The grains contaminated with Cd (soaked in CdCI 2solution) were prepared prior to the experiment. The AAS analysis of the grains revealed that actual levels of Cd were between 90 and 95% of the intended level. The same analysis showed that the grains contained 60-80 mg Fe/kg, 4-5 mg Cu/ kg, and 20-25 mg Zrdkg DM. In addition, an identical amount of

Cadmium and lipid peroxidation apple was offered to all animals (3 g/vole/week) and was eaten completely. The food intake was measured weekly.

77

gestion at room temperature, 72% perchloric acid (0.5 ml) was added and the mixture was heated at 100°C for 3 h. Finally, the temperature was raised to 150°C and digestion continued for

Assays

another 4 h. Deionized water was added to the residue (about 0.3 ml) after digestion to a volume of 3.0 ml. The concentrations

At the end of the 6-week exposure period, the bank voles

of Fe, Cu and Zn were determined by AAS in an air-acetylene

were weighed, euthenized with ether, and the liver and both kid-

flame, whereas Cd analyses were carried out by electrothermal

neys were removed. About 0.5 g of the fresh liver and kidneys

AAS using an AAS 3 Carl Zeiss instrument with an EA3 fur-

(pooled from two voles) were transferred to 4.5 ml of chilled

nace attachment. Samples of bovine liver 1577b (National Insti-

0.25 M sucrose solution and homogenized with a Teflon pestle

tute of Standards and Technology, Gaithersburg, MD) and CL- 1

in a glass homogenizer. Aliquots (0.2 and 3.0 ml) of the ho-

cabbage leaves (AGH, Poland) were also analysed in an identi-

mogenate were taken for determination of LPO and metal lev-

cal manner to check accuracy of the method. The recovery of

els, respectively. The remaining homogenate was centrifuged at

Fe, Cu, Zn and Cd amounted to 90-95%.

20.000 g for 20 min at 4°C, and the resulting supernatant was

Statistical analysis

removed for MT assay. LPO was assessed by measuring malondialdehyde (MDA) formation, using the thiobarbituric acid (TBA) assay (17).

Results were reported as mean + SD. The data were ana-

Tetraethoxypropane was used to prepare a calibration curve.

lysed by one- and two-way analysis of variance (ANOVA). Dif-

The results were expressed as TBA-RS (nmol/g FM).

ferences between means were determined by Duncan's multi-

MT content was determined by a Cd-saturation method

ple-range test. Differences at p < 0.05 were considered statisti-

adapted from (18). Briefly, in a 1.5 ml vial 0.1 ml of sample was

cally significant. The relationship between LPO and the trace

incubated for 10 min at room temperature with 1.0 ml Tris-HC1

element level in the liver and kidneys was evaluated by using

buffer (0.03 M, pH=7.8) containing 1.0 Bg Cd/ml as CdC12. To

simple and multiple regression analyses. All analyses were per-

remove non-MT-bound Cd, bovine hemoglobin (Sigma) (0.1 ml

formed on natural log-transformed data.

of a 5% solution in H20) was added and the sample was heated for 1.5 min at 100°C, then cooled and centrifuged for 5 min. at

Results

10.000 g. Addition of hemoglobin, heating and centrifugation of the sample were repeated three times. The MT was calculated from the Cd content in the resulting clear supernatant using a

The 6-week exposure to dietary Cd at amounts of 40 and 80

molecular weight of 6600 and the definite molar ratio of 7

mg/kg affected neither the consumption of food (2.0-2.5 g per

moles of Cd per mole of MT.

day), nor the final body and organ weights of the male and fe-

Metal determinations were performed as described previ-

male bank voles. The mean body weights of the control, Cd-40

ously (16). The homogenate (3.0 ml) was placed in a glass tube

and Cd-80 males were 16.0 + 2.0, 15.7 + 1.8 and 15.8 + 2.2 g,

with 1.5 ml of concentrated nitric acid. After 20 h of sample di-

respectively. The liver and kidney weights were 801 + 90 and

Table 1. Lipid peroxidation (TBA-RS), trace elements and metallothionein (MT) levels in the liver of bank voles exposed for 6 weeks to dietary Cd Dietary Cd (mg/kg DM)

TBA-RS (nmol/g FM)

Iron (mg/kg FM)

Copper (mg/kg FM)

Zinc (mg/kg FM)

Cadmium (mg/kg FM)

MT (mg/kg FM)

Males Control 40 80

217 _+50 a 59+15 b 38 -+ 10°

559 ± 240 ~ 108+ 37 b 61 -+ 17c

10.3 _+3.@ 6.6+2.0 b 6.0 4- 1.7b

38.2 + 5.5 a 40.6±5.2 a 39.8 4- 2.3 a

0.22 _+ 0.05 a 14.7-+ 3.5 b 37.3 -+ 10c

14.6 + 4.5 ~ 119+25 b 187 -+ 37 °

Females Control 40 80

185+52 ~ 103 -+ 25 ~ 105 -+ 17d

597_+135" 242 -+ 97 d 147 -+ 52 bd

40.6+5.3 a 38.0 __5.3 a 42.3 _+5.0 a

0.18+0.03 ~ 14.8 -+ 2.8 b 41.2 + 9.5 c

11.5-+ 3.3 a 116 + 20 b 194 _+40 °

Source of variation

Two-way ANOVA - p values

Cd Sex Cd x Sex

0.0000 0.016 0.0005

NS NS NS

0.0000 NS NS

0.0000 NS NS

0.0000 0.0025 0.021

9.1+2.7 ab 8.7 4- 1.5 ~b 8.9 -+ 1.7 ~b

0.0276 NS 0.0272

Values represent the mean + SD, n = 8. Means in the same column marked with a different superscript letter are significantly different at p < 0.05. NS: not significant.

78

T. Wlostowski, A. Krasowska and B. Godlewska-Zylkiewicz

Table 2. Lipid peroxidation (TBA-RS), trace elements and metallothionein (MT) levels in the kidneys of bank voles exposed for 6 weeks to dietary Cd Dietary Cd (mg/kg DM)

TBA-RS (nmol/g FM)

Iron (mg/kg FM)

Copper (mg/kg FM)

Zinc (mg/kg FM)

Cadmium (mg/kg FM)

MT (mg/kg FM)

Control 40 80

194 _+40 a 183 _+35 a 128 4- 27 b

168 +_32 ~ 148 + 25 a 97 _ 20 b

8.4 _+2.2 a 8.4 + 2.1 a 7.9 _+ 1.5 a

35.2 4- 6.5 a 36.3 + 6.0 a 37.3 _+4.5 a

0.42 _+0.14 a 17.0 + 3.3 b 36.0 _+6.9 c

20.3 + 4 ~ 140 _ 30 b 239 + 40 ~

Values represent the mean + SD for 8 samples. The kidneys of two bank voles were pooled and used as one sample. There were no significant differences between males and females and therefore they were combined and presented jointly. Means in the same column marked with a different superscript letter are significantly different at p < 0.05. 180 + I1 mg, 795 + 75 and 172 + 10 mg, and 796 + 77 and 173

group were found only in the Cd-80 bank voles. Renal Cu and

+ 12 mg for the control, Cd-40 and Cd-80 male bank voles, re-

Zn were not influenced by dietary Cd, while the concentrations

spectively. There were no significant differences in the body

of Cd and MT in the kidneys appeared to increase significantly

and organ weights between male and female bank voles.

in a dose-dependent fashion.

As can be seen in Table 1, TBA-RS formation (an index of

Simple regression analysis showed a positive correlation

LPO) in the liver was affected significantly by dietary Cd, the

between TBA-RS, and Fe and Cu concentrations in the liver of

sex of the animals and the interaction between these two. Die-

bank voles (comparisons 1 and 2) (Table 3). The same analysis

tary Cd decreased TBA-RS level in the liver of both male and

revealed an inverse relation between hepatic MT and TBA-RS

female bank voles but the effect was dose-dependent only in the

(comparison 3). Multiple regression analysis confirmed that the

males. The extent of the decrease in the hepatic LPO was also

hepatic Fe and Cu, but not M T correlated significantly with

more pronounced in the males than females: 3.7-5.7 - and 1.7-

TBA-RS (comparison 4). The two regression analyses showed

fold, respectively. The changes in the hepatic LPO paralleled

that in the kidneys TBA-RS correlated significantly only with

closely those of the iron concentrations (Table 1). Dietary Cd

the Fe concentration (Table 3).

brought about a depletion in the hepatic Fe in a dose-dependent manner; however, compared to control animals the males appeared to be more sensitive than females to Cd-induced Fe de-

Discussion

pletion. In contrast to TBA-RS and Fe, dietary Cd induced a decrease in hepatic Cu only in males in a dose-independent

The present study demonstrated a drop in the hepatic and re-

fashion and had no influence on Zn concentrations; regardless

nal LPO below the control value in bank voles exposed

of sex, the liver Cd and MT levels increased significantly in a

subchronically to elevated levels of dietary Cd. Thus, our re-

dose-dependent pattern (Table 1).

sults contrats with those found in the liver and kidneys of Cd-

Two-way ANOVA revealed that the kidney TBA-RS, Fe,

injected animals (2, 3, 4). Those studies have shown that Cd en-

Cu, Zn, Cd and MT levels were not affected by the sex of the

hances hepatic and renal LPO and that the process is related to

animal; therefore, the data obtained for the male and female

an accumulation of reactive oxygen species (ROS) in the pres-

bank voles were combined and presented jointly (Table 2). Di-

ence of elevated doses of the metal, which probably inhibits cel-

etary Cd decreased renal TBA-RS and Fe levels in a dose-de-

lular detoxifying enzymes such as catalase and superoxide dis-

pendent manner but significant differences from the control

mutase (19). In light of those studies it is difficult to imagine

Table 3. Relationship of lipid peroxidation (TBA-RS) in the liver and kidneys to hepatic or renal concentrations of iron, copper and metallothionein (MT) in the bank vole under study No.

Comparison

Liver l. TBA-RS 2. TBA-RS 3. TBA-RS 4. TBA-RS Kidneys 5. TBA-RS 6. TBA-RS 7. TBA-RS 8. TBA-RS

versus Fe versus Cu versus MT versus Fe, Cu, MT versus versus versus versus

Fe Cu MT Fe, Cu, MT

n

Regression equation

Coefficient of correlation

p value

48 48 48 48

In TBA-RS In TBA-RS In TBA-RS In TBA-RS

1.15 + 0.64 In Fe 2.15 + 1.1 In Cu 6.06 - 0.36 In MT 0.52 In Fe + 0.61 In Cu - 0.02 In MT + 0.65 (p=0.0001) (p=0.002) (p=0.75)

0.82 0.59 -0.63

0.0000 0.009 0.008

0.87

0.0000

= 2.2 + 0.59 In Fe = 5. I + 0.01 In Cu = 5.48 - 0.08 In MT = 0.65 In Fe + 0.16 In Cu + 0.01 In MT + 1.5 (p=0.0001) (p=0.33) (p=0.8)

0.70 0.00 -0.20

0.0006 0.96 0.23

0.62

0.002

24 24 24 24

in TBA-RS in TBA-RS in TBA-RS in TBA-RS

= = = =

In brackets p values for the particular regression coefficients are presented, ln: natural Iogarithm.

Cadmium and lipid peroxidation that in the liver and kidneys of bank voles Cd ions, instead of

79

by us in the liver and kidneys of bank voles fed diet for 6 weeks

inhibiting antioxidant protection mechanisms could activate

containing 80 mg Cd/kg (26). Other authors have also contested

them, thereby decreasing ROS accumulation and LPO. Therefore, the intracellular Cd, by itself was probably not responsible

the main role of LPO in cell injury induced by Cd and other toxicants (1, 27, 28). Thus, another mechanism is probably in-

for the reduction of LPO in the bank vole.

volved in Cd-induced tissue injury.

Human and animal studies have demonstrated that dietary Cd decreases intestinal iron absorption and its concentration in

In conclusion, the data of the present study suggest that die-

the liver and kidneys (14, 15, 20). As Fe plays an essential role

tary Cd decreases hepatic and renal LPO indirectly, through lowering the tissue iron concentration. These results also sug-

in the cellular oxidative processes, one would expect major

gest that Fe is probably a main determinant of LPO under phys-

changes to occur concurrently in the tissue ROS formation and LPO. Indeed, dietary Cd brought about a parallel decrease in the

iological conditions.

hepatic and renal Fe and TBA-RS concentrations in the bank vole (Table 1 and 2). These data suggest that dietary Cd de-

Acknowledgement

creased hepatic and renal LPO indirectly through changes in iron metabolism. The idea is in good agreement with other studies showing a close correlation .between tissue Fe and LPO (9, 10, 13). However, the exact mechanism by which Cd-induced

These studies were supported by a grant from the Poland State Committee for Scientific Research (6-PO4C-048-10).

Fe deprivation affects hepatic and renal LPO in the bank vole is uncertain and requires further study. Nevertheless, there may be at least two possible mechanisms which are not mutually exclusive: (a) Fe catalyses in a concentration-dependent manner the production of hydroxyl radicals (-OH) in the Fenton reaction (21) and the radicals induce LPO also in a concentration-dependent fashion; and (b) Fe, as a component of several proteins and enzymes composing the mitochondrial respiratory chain, is responsible for the tissue respiration capacity. Thus, a steep decrease in the tissue Fe, such as that observed in the present study, may lead to disturbances in cellular respiration (e.g. a decrease in oxygen consumption) and, in consequence, to the reduction in ROS production and lipid peroxidation. It is well known that besides iron, copper, zinc and metallothionein as well play an essential role in LPO; Zn and MT are known to protect fatty acids from peroxidation by inhibiting the production of or by scavanging ROS (11, 22), whereas free Cu ions can enhance LPO (23). The present study showed that dietary Cd did not change hepatic and renal Zn levels; in addition, Cu and MT concentrations in the liver of female bank voles and in the kidneys of both sexes did not correlate significantly with TBA-RS (Table 1, 2 and 3). This suggests that Cu, Zn and MT were probably not responsible for the reduction of LPO induced by the dietary Cd. However, our results do not exclude the possibility that in the liver of male bank voles a decline in Cu might be involved in some way in the inhibition of LPO. Finally, it is worth noting that in contrast to dietary Cd (Table 1 and 2), the injection of CdC12 causes an increase in the hepatic and renal Cu (25), which may account, at least in part, for a rise in LPO under such conditions (2, 3, 4). Whether or not injected Cd also effects the tissue Fe is not known at present and remains to be elucidated. Enhanced LPO has been considered to play an important role in Cd-induced liver and kidney injury (2, 24, 25). The results obtained in the present study indicate, however, that LPO cannot be responsible for histopathological changes observed

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