Adverse Effects of Combined Arsenic and Fluoride on Liver and Kidney in Rats

Fluoride Vol. 32 No. 4 243-247 1999 Research Report 243

ADVERSE EFFECTS OF COMBINED ARSENIC AND FLUORIDE ON LIVER AND KIDNEY IN RATS

Kai-tai Liu,a Guo-Quan Wang, Li-Ying Ma, Ping jang, Bi-Yu Xiao, Chen Zhang Urumqi, Xinjiang, China

SUMMARY: In a subacute animal study, the effects of arsenic and fluoride on liver and kidney in rats were investigated. The results indicated that arsenic, fluoride and their combination affected the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the contents of malondialdehyde (MDA) and sulphhydryl groups (-SH). Antagonistic effects were found between arsenic and fluoride as well as on Zn, Fe, Ca, and Mg in liver and Ca, Mg, Sr, and Al in kidney. Arsenic significantly increased the liver and kidney content of Fe. For Mn there seemed to be synergism between arsenic and fluoride. The effects of arsenic and fluoride on liver and kidney have two aspects: one is direct action; the other is indirect – disturbances of free radical balance and abnormal metabolism of some inorganic elements. Keywords: Antioxidases, Arsenic-fluoride poisoning, Arsenism, Lipid peroxidation, Metal- lic elements, Rat liver, Rat kidney.

INTRODUCTION Combined arsenic-fluoride poisoning is an exceptional disease in the world. Since the 1960s, a large number of wells have been dug due to lack of water in the Kuitun area of China. Unfortunately, this water is abundant in fluoride as well as arsenic. After the 1970s, endemic fluorosis and arsenism ap- peared among the residents in some parts of the area, involving about 50,000 people and an area of 1200 square kilometers. Both domestic and foreign sci- entific investigators have studied arsenism and fluorosis, and the results indi- cate that both arsenic and fluoride are able to cause injury to liver and kidney.1-3 But studies of the combined effects of fluoride and arsenic on liver and kidney, especially in low dose and long-term contact conditions have not been reported. In recent years areas of arsenic-fluoride poisoning have been succes- sively discovered in the provinces of Neimeng and Gueizhou besides Xin- jiang. It is therefore highly important to investigate the pattern and mechanism of combined arsenic and fluoride injury on liver and kidney. With the further investigation on the theory of free radicals, it has been found that free radicals are new pathogenic factors which induce damage to lipid peroxidation. Simultaneously they might be the original adverse reac- tions from toxicants. Under normal conditions, the level of free radical pro- duction and elimination keeps a dynamic balance, and the metabolism of metallic elements maintains the relative stability. In order to investigate the effects of arsenic and fluoride together on lipid peroxidation and antioxidant systems and the metabolism of metallic elements, the effects of arsenic and fluoride on MDA, SOD, GSH-Px, -SH and nine metallic elements on liver and kidney were studied by means of a subacute animal experiment.

——————————————— aFor correspondence: Department of Environmental Hygiene, Xinjiang Medical Universi- ty, Urumqi, Xinjiang, China 830054. 244 Liu, Wang, Ma, Jang, Xiao, Zhang

MATERIALS AND METHODS Fifty six healthy Wistar rats (NO.0000569) weighing 140-160 g at the be- ginning of the experiment were obtained from the Laboratory Animal Center of Shanghai Medical University. The rats were randomly divided into four equal groups of 14 animals each: F group, As group, F+As group, and the control group. All rats were housed in the same room with free access to food and water. The control group was given distilled water in which the fluoride and arsenic concentration was 0 mg/L. The experimental groups were given drinking water containing 150 mg/L sodium fluoride (NaF), 75 mg/L arsenic trioxide (As2O3), and 150 mg/L NaF + 75 mg/L As2O3, respectively. After six months the rats were killed. The hepatic and renal tissues (ap- proximately 0.3 g each) were quickly removed, weighed, homogenized, and centrifuged. The supernatant was then assayed for the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the contents of malondialdehyde (MDA) and sulphhydryl groups (-SH).4-7 Other hepatic and renal tissue were baked at 80°C, and then assayed with an “Inductively Coupled Plasma Atomic Emission Spectrometer” (ICP-AES) for metallic elements (Cu, Zn, Fe, Cr, Pb, Sr, Al, Mg, and Ca). Calculated values of experimental groups are reported as mean ± s.e. The significance of the difference between means at the same period was deter- mined by analysis of variance. RESULTS Hepatic tissue: Antioxdase levels (SOD, GSH-Px and -SH) decreased significantly by the sixth month in the groups of rats given water containing either 150 mg/L NaF or 75 mg/L As2O3. They also decreased in the 150 mg/L NaF plus 75 mg/L As2O3 group, but were not significantly different from the controls (Table 1). The content of Zn, Fe, Ca, and Mg in the liver in the NaF group and the As2O3 group were significantly different from the controls. The content of Mg in the NaF + As2O3 group was lower than in the control group. In this study, other elements were not significantly different among the four groups (Tables 2 and 3).

Table 1. Hepatic tissue SOD, GSH-Px activities, and MDA, -SH concentrations Group MDA SOD GSH-Px -SH (nmol/mgpro) (U/mgpro) (U/mgpro) (g/mgpro) Control 7.40 ± 3.68 122.81 ± 26.32 386.40 ± 74.48 79.13 ± 13.64 NaF 9.99 ± 2.37 98.25 ± 18.53ad 246.57 ± 39.46bd 48.25 ± 8.92ad ac bd bd As2O3 6.47 ± 2.48 99.83 ± 7.56 241.12 ± 39.64 49.51 ± 6.27

NaF+As2O3 7.88 ± 3.08 120.30 ± 17.56 382.28 ± 65.49 66.96 ± 18.93 Note: Compared with Control group - a: P<0.05 b: P<0.01 Compared with NaF + As2O3 group - c: P<0.05 d: P<0.01

Renal tissue: Lipid peroxidation level increased in the As2O3 group, but antiox- idase levels were lower than in the control group (Table 4). The content of Fe

Fluoride 32 (4) 1999 Adverse effects of arsenic and fluoride on liver and kidney in rats 245

in the NaF + As2O3 group was highest among all 4 groups, and Cr was lowest in the NaF group (Table 5). The contents of Mg, Ca, Sr and Al in the NaF and As2O3 groups were significantly lower than in the control and the NaF + As2O3 groups (Table 6).

Table 2. Hepatic tissue Zn, Ca, Mg, and Fe concentrations (g/g) Group Zn Ca Mg Fe Control 280.43 ± 143.22 826.38 ± 207.91 521.25 ± 44.17 710.44 ± 300.85 NaF 225.89 ± 81.43f 519.13 ± 140.06bc 394.17 ± 49.79bd 791.91 ± 255.32f bd a b bd As2O3 745.29 ± 278.06 629.63 ± 140.02 420.31 ± 40.76 1315.47 ± 409.90

NaF+As2O3 247.01 ± 131.62 771.60 ± 149.97 470.73 ± 53.99b 898.65 ± 280.68 Note: Compared with Control group - a: P<0.05 b: P<0.01 Compared with NaF + As2O3 group - c: P<0.05 d: P<0.01 Compared with As2O3 group - f: P<0.01

Table 3. Hepatic tissue Mn, Cu, Sr, Pb, and Cr concentrations (g/g) Group Mn Cu Sr Pb Cr Control 2.06 ± 0.86 11.17 ± 5.51 0.80 ± 0.29 5.99 ± 2.81 7.49 ± 3.98 NaF 1.85 ± 0.24 9.01 ± 1.38 0.56 ± 0.08 4.95 ± 2.25 6.25 ± 2.05

As2O3 3.27 ± 1.83 11.40 ± 4.10 0.75 ± 0.31 7.05 ± 2.98 10.11 ± 5.59

NaF+As2O3 2.51 ± 2.11 9.20 ± 5.08 0.68 ± 0.14 6.10 ± 2.31 8.70 ± 3.71 Note: Comparison among the four groups P<0.05

Table 4. Renal tissue SOD, GSH-Px activities, and MDA, -SH concentrations Group MDA GSH-Px -SH SOD (nmol/mgpro) (U/mgpro) (g/mgpro) (U/mgpro) Control 3.16 ± 1.45 92.46 ± 26.47 454.41 ± 92.16 68.82 ± 17.96 NaF 4.46 ± 1.32 58.76 ± 24.93b 327.32 ± 111.78ae 43.84 ± 8.33b a b bd bc As2O3 5.11 ± 1.23 41.79 ± 18.07 243.14 ± 54.95 39.80 ± 9.05 b b NaF+As2O3 4.24 ± 0.74 50.54 ± 26.27 399.37 ± 72.76 51.56 ± 12.02 Note: Compared with Control group - a: P<0.05 b: P<0.01 Compared with NaF + As2O3 group - c: P<0.05 d: P<0.01 Compared with As2O3 group - e: P<0.05

Table 5. Renal tissue Fe, Mn, Cr, Zn, and Pb concentrations (g/g) Group Fe Mn Cr Zn Pb Control 623.97 ± 260.25 1.25 ± 0.38 10.58 ± 5.35 372.96 ± 184.40 10.66 ± 6.07 NaF 584.68 ± 280.83df 0.98 ± 0.24d 4.89 ± 2.57ade 404.75 ± 245.34 10.71 ± 3.93 b As2O3 917.52 ± 258.67 1.33 ± 0.61 10.97 ± 6.43 398.28 ± 163.71 14.99 ± 8.56 b be NaF+As2O3 1056.25 ± 130.23 2.42 ± 1.19 14.32 ± 5.62 570.69 ± 203.42 16.80 ± 9.12 Note: Compared with Control group - a: P<0.05 b: P<0.01 Compared with NaF + As2O3 group - c: P<0.05 d: P<0.01 Compared with As2O3 group - e: P<0.05 f: P<0.01

Fluoride 32 (4) 1999 246 Liu, Wang, Ma, Jang, Xiao, Zhang

Table 6. Renal tissue Mg, Ca, Sr, and Al concentrations (g/g) Group Mg Ca Sr Al Control 456.06 ± 80.60 942.32 ± 184.38 1.13 ± 0.18 142.63 ± 47.95 NaF 338.46 ± 40.27bc 650.38 ± 121.71bd 0.91 ± 0.26bd 72.71 ± 20.87bd bc bd bd bd As2O3 323.05 ± 48.08 688.32 ± 66.04 0.87 ± 0.17 78.37 ± 24.55

NaF+As2O3 414.12 ± 66.79 954.55 ± 155.59 1.22 ± 0.18 144.14 ± 54.18 Note: Compared with Control group - b: P<0.01 Compared with NaF + As2O3 group - c: P<0.05 d: P<0.01

DISCUSSION Activities of many enzymes and the levels of inorganic elements are important factors for keeping in good health. When they become abnormal, the structure and function of internal organs of the body can be disturbed. It has been reported that fluoride and arsenic cause injury to liver and kidney.8-11 By using the method of subacute animal experimentation, we have examined the effects of arsenic, fluoride, and arsenic plus fluoride on liver and kidney of rats. Our findings can be summarized as follows: Effects on liver: Arsenic, fluoride, and their combination affected the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the contents of malondialdehyde (MDA) and sulphhydryl groups (-SH), and of Zn, Fe, Ca and Mg. There were antagonistic effects on antioxidation between arsenic and fluoride. Due to this antagonism between the effects of arsenic plus fluoride, Ca and Mg in rat liver were lower than from the effects of either one when used alone. Effects on kidney: Arsenic increased the contents of malondialdehyde (MDA) and caused accumulation of Fe. In its effect on Cr, arsenic reduced the action of fluoride. Effects of the combination of arsenic and fluoride on SOD, GSH- Px and -SH in rat kidney were greater than those of arsenic or fluoride alone. The combined action of arsenic and fluoride increased the content of Mn. In this respect, there was synergism between arsenic and fluoride. As indicated above, the toxicity of arsenic and fluoride in liver and kidney has two aspects. One is the direct action. The other is indirect action – the dis- turbance of free radical balance and abnormal metabolism of some inorganic elements. In view of the antagonism found in many indices in liver and kidney, cell metabolism processes were affected. As a result, normal metabolism of cells must have been disturbed with injury to liver and kidney.

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—————————————————————— Published by the International Society for Fluoride Research Editorial Office: 17 Pioneer Crescent, Dunedin 9001, New Zealand

Fluoride 32 (4) 1999