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Cataractogenic Potential of Bromate-Mediated Oxidative Stress in Rabbits



N.P. Okolie and J.C. Ikewuchi
 
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ABSTRACT

The effects of bromate-induced oxidative stress on some cataractogenic indices were studied in the lens, cornea and retina of New Zealand White rabbits given 60 mg kBrO3/kg body wt/day by gavage for 28 days using gravimetric, enzymatic, colorimetric and spectrophotometric procedures. The bromate treatment led to significant decreases in the activities of Na+-K+-ATPase in the lens and cornea and significant increases in corneal catalase and lenticular superoxide dismutase, SOD activities (p<0.05). However the retinal enzymes were unaffected. Significant reductions in the antioxidant vitamins A and C were accompanied by significant elevation of malondialdehyde, MDA in all the tissues investigated (p<0.05). These results are strongly indicative of bromate-induced oxidative stress. The elevation of SOD and catalase activities appear to be adaptive responses to cope with this challenge. The implication of these findings for cataractogenesis and the likelihood of existence of tissue-specific reactive species in bromate-induced oxidative stress in ocular tissues are discussed.

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  How to cite this article:

N.P. Okolie and J.C. Ikewuchi , 2004. Cataractogenic Potential of Bromate-Mediated Oxidative Stress in Rabbits. Journal of Medical Sciences, 4: 158-163.

DOI: 10.3923/jms.2004.158.163

URL: https://scialert.net/abstract/?doi=jms.2004.158.163

INTRODUCTION

Potassium bromate, KBrO3 is an oxidizing agent employed widely as a maturing agent in flour and a dough conditioner in the bakery industry[1,2] Bromate is a constituent of cold-wave hair formulations and may be present as a contaminant in drinking water due to the conversion of bromides to bromates during ozonation of raw water[3]. Several toxic effects of KBrO3, mainly carcinogenic and mutagenic effects have been reported in experimental animals[4-8]. Lethal oral doses of bromate in humans have been estimated as 154-385 mg kg-1 body wt, while serious poisoning results at doses of 46-92 mg kg-1 body wt[9]. Oral doses of 185-385 mg kg-1 body wt result in irreversible toxic effects like renal failure and deafness in humans, while lower doses are associated with vomiting, diarrhoea, nausea and abdominal pain[9]. Due to the harmful effects of KBrO3, the Joint Expert Committee on Food Additives, JECFA has recommended that when bromate is used in food processing no residues should be left[10]. However, we recently reported toxic bromate residues in Nigerian bread[11], which shows that humans are still being exposed to this toxin notwithstanding several existing legislations outlawing its use.

Despite the plethora of evidence on toxicity of KBrO3 in human and animal tissues, there is a dearth of information on its effect on ocular function. It is known that bromate induces oxidative stress in tissues[2,3,7]. Indeed oxidative DNA damage appears to be the basis of bromate-induced kidney carcinogenesis[7,12,13]. Evidence abound suggesting a direct causal relationship between oxidative stress and cataractogenesis[14-16]. This study was therefore aimed at ascertaining the effect of KBrO3 toxicity on some rabbit ocular tissues and their potential for cataractogenesis in this animal model.

MATERIALS AND METHODS

Animals and treatment: Three month old New Zealand White rabbits [initial mean weight =1.40 kg ] were obtained from a breeder in Benin City. The animals were housed singly in clean, disinfected metal hutches and acclimatized on guinea growers mash (product of Bendel Feed and Flour Mills, BFFM Ltd, Ewu, Nigeria] for 2 weeks prior to the experiment. They were subsequently weighed and randomly assigned to 2 groups (5 per group). Members of one groups (test) were given 60 mg KBrO3 /kg body wt /day for 28 days. The bromate was dissolved in physiological saline and administered by gavage. Members of the other group (control) received saline (by the same route) in place of bromate. Both groups were fed ad libitum on mash and clean drinking water was liberally provided. Fresh feed was provided daily, while stale remnants were weighed and discarded. The rabbits in the test group were weighed weekly to allow for adjustments in bromate. At the end of 28 days, all animals were weighed and sacrificed painlessly by decapitation under chloroform anesthesia. The eye balls were rapidly excised and the lens, cornea and retina were quickly dissected out. Each tissue was ground thoroughly in a hand mortar with some grains of acid-washed sand and 5.0 ml of ice-cold physiological saline for 10 min. The resultant tissue homogenates were kept at 4°C and used for subsequent assays. All homogenates were analyzed within a few hours of preparation.

Enzyme and metabolite assays: Catalase was assayed spectrophometrically by estimating the rate of decomposition of hydrogen peroxide, H2O2[17]. SOD was assayed by monitoring the inhibitory effect of the enzyme on the auto-oxidation of epinephrine[18].

The assay of Na+-K+-ATPase was carried out colorimetrically by estimating the amount of inorganic phosphate, Pi liberated upon incubation of the tissue homogenates with disodium ATP[19]. The Pi was determined colorimetrically using ammonium molybdate[20]. MDA was estimated colorimetrically based on its reaction with thiobarbiturate to yield a pink-coloured complex that absorbs strongly at 532 nm[21].

Ascorbic acid was assayed in a colorimetric reaction using 2,4-dinitrophenyl hydrazine, 2,4-DNP[22]. In this assay, ascorbate reacts with 2,4-DNP to give a red osazone which absorbs strongly at 540 nm.

Vitamin A assay involved the precipitation of proteins with absolute ethanol followed by extraction of tocopherols and carotenes with petroleum ether (60-80°C) and estimation of the carotenes by their absorbance at 459 nm[23].

Statistics: The Means±SEM of the values of the various parameters for the test and control groups were analyzed for statistically significant differences using students t-test.

RESULTS

Feed intake and weight gain were significantly lower (p<0.05) in the bromate-treated rabbits when compared to controls (Table 1). Catalase activity was significantly increased (p<0.05) in the cornea of the rabbits given KBrO3, while the enzyme was virtually unaffected in the lens and retina (Fig. 1). The bromate treatment led to significant increase (p<0.05) in lenticular SOD activity, while the corneal and retinal enzymes were relatively unchanged (Fig. 2).

Table 1: Feed intake and weight changes for rabbits in the test and control groups
Image for - Cataractogenic Potential of Bromate-Mediated Oxidative Stress in Rabbits
Values are Mean±SEM (n=5). For each parameter, those having different superscript across differ significantly (p<0.05)

Image for - Cataractogenic Potential of Bromate-Mediated Oxidative Stress in Rabbits
Fig. 1: Catalase activities in the lens, cornea and retina of KBrO3-treated rabbits and controls (*p<0.05 compared to control, n=5 per group)

Image for - Cataractogenic Potential of Bromate-Mediated Oxidative Stress in Rabbits
Fig. 2: SOD activities in the lens, cornea and retina of KBrO3-treated rabbits and control (*p<0.05 compared to control, n=5 per group)

Image for - Cataractogenic Potential of Bromate-Mediated Oxidative Stress in Rabbits
Fig. 3: Na+-K+-ATpase activities in the lens, cornea and retina of KBrO3-treated rabbits and controls (*p<0.05 compared to control, n=5 per group)

Image for - Cataractogenic Potential of Bromate-Mediated Oxidative Stress in Rabbits
Fig. 4: MDA leaves in the lens, cornea and retina of KBrO3-treated rabbits and controls, n=5 per group)

Na+-K+-ATPase activity was significantly decreased in the lens and cornea (Fig. 3), while significant elevation of MDA, accompanied by significant decreases in the levels of vitamins A and C were obtained in all the tissues studied (Fig. 4, 5 and 6, respectively).

DISCUSSION

Most of the available data on acute toxicity studies on bromate refer to rats, mice and hamsters[6,24,25].

Image for - Cataractogenic Potential of Bromate-Mediated Oxidative Stress in Rabbits
Fig. 5: Ascorbic acid levels in the lens, cornea and retina of KBrO3-treated rabbits and controls (*p<0.05 compared to control, n=5 per group)

Image for - Cataractogenic Potential of Bromate-Mediated Oxidative Stress in Rabbits
Fig. 6: Vitamin A levels in the lens, cornea and retina of KBrO3-treated rabbits and controls (*p<0.05 compared to control, n=5 per group)

The LD50 of orally-administered KBrO3 in mice is 223-363 mg kg-1 body wt[26], while LD50 of 280-495 mg kg-1 body wt have been reported for rats[24]. Based on various tissue toxicity effects, the least observable adverse effect levels, LOAEL of bromate in rats are 6.1-30 mg kg-1 body weight/day[27-29]. However there is a dearth of information regarding bromate toxicity studies involving rabbits. Prior to the present study, preliminary exposure trials revealed that a dose of 120 mg kg-1 body weight/ day was lethal to rabbits and resulted in mortalities within 5 days. On the other hand, when this dose was halved to 60 mg kg-1 body wt/day, it was well tolerated without any obvious adverse effects. The observed significant decrease in weight gain for the KBrO3-treated rabbits is probably a consequence of depressed feed intake due to the toxic effects of bromate. Several workers have reported similar bromate-induced decreases in body weight gain in rats[24,27,29,30].

Increases in blood and tissue levels of thiobarbituric acid-reactive substances, mainly MDA, are very reliable indices of oxidative stress and lipid peroxidation[31]. Thus the profiles of MDA and antioxidant vitamins in the bromate-treated rabbits clearly indicate that bromate provokes oxidative stress in ocular tissues. Several reactive oxygen species, ROS are derivable from bromate viz., peroxynitrile, ONOO¯, hydrogen peroxide, H2O2, superoxide anion, O2¯ and hydroxyl radical, OH-[12]. The elevation of corneal catalase activity in the bromate-treated rabbits is most likely an adaptive response to cope with the challenges of raised H2O2 levels in this tissue. Similarly, adaptive response may account for the elevation of SOD in the lens, which implies that in this tissue O2¯ rather than H2O2 might be the principal agent for bromate-provoked oxidative damage. In contrast, retinal SOD and catalase were practically unaffected by KBrO3 not with standing the MDA and antioxidant vitamin profiles in this tissue. Thus it is likely that a ROS different from O2¯ and H2O2, may be implicated in bromate-induced oxidative stress in the retina. The involvement of tissue-specific ROS in bromate-mediated ocular damage is in agreement with the finding that SOD and catalase have no inhibitory effects on bromate-mediated renal damage[32]. Indeed studies have revealed that bromine oxides and bromine radicals, rather than H2O2, are responsible for the oxidative stress and renal tumours induced in rat by KBrO3[5,12,30,32].

Cataracts is the final stage of a degenerative ocular disease resulting in opacification of the lens. Evidence abound linking ocular oxidative stress to cataractogenesis[14-16]. Thus cataracts may result if ocular antioxidant status is compromised[14,34]. Ocular oxidative stress leads to changes in electrolyte balance in the lens and cornea, as a consequence of depression of Na+-K+-ATPase activity in these tissues. Na+-K+-ATPase functions to maintain electrolyte balance in the lens and cornea[35]. The enzyme is inhibited in cataractous lens[36,37] and by cataractogenic factors such as free radicals[28,39], H2O2[40,41] and changes in membrane lipid composition[42]. Consequently the pattern of Na+-K+-ATPase and other antioxidant parameters in the lens and cornea of the KBrO3 toxified rabbits are strongly indicative of the cataractogenic potential of bromate. Thus of animal-to man extrapolation is allowed, our results suggest that aside from its well established carcinogenic and mutagenic effects, bromate may be a strong predisposing factor in cactaractogensis.

REFERENCES

1:  Chipman, J.K., J.E. Davies, J.L. Parsons, J.O. Neill and J.K. Fawell, 1998. DNA oxidation by potassium bromate: A direct mechanism or linked to lipid peroxidation? Toxicology, 126: 93-102.

2:  Ueno, H., K. Oishi, Y. Sayato and K. Nakamuno, 2000. Oxidative cell damage in Kat-Sod assay of oxyhalides as inorganic disinfection byproducts and their occurrence by ozonation. Arch. Environ. Contam. Toxicol., 38: 1-6.
CrossRef  |  PubMed  |  Direct Link  |  

3:  Umemura, T., A. Takagi, K. Sai, R. Hasegawa and Y. Kurokawa, 1998. Oxidative DNA damage and cell proliferation in kidneys of male and female rats during 13 weeks exposure to potassium bromate (KbrO3). Arch. Toxicol., 5: 264-269.

4:  Kurokawa, Y., N. Takamura, C. Matsuoka, T. Imazawa, Y. Matsushima, H. Onedera and Y. Hayashi, 1987. Comparative studies on lipid peroxidation in the kidney of rats, mice and hamsters and on the effects of cysteine, glutathione and diethyl maleate treatment on mortality and nephrotoxicity after administration of potassium bromate. J. Am. Coll. Toxicol., 6: 489-501.

5:  Sai, K., A. Takagi, T. Umemura, R. Hasegawa and Y. Kurokawa, 1991. Relation of 8-hydroxydeoxyguanosine formation in rat kidney to lipid peroxidation, glutathione level and relative organ weight after a single administration of potassium bromate. Jap. J. Cancer Res., 82: 165-169.
CrossRef  |  

6:  Giri, U., M. Igbal and M. Athar, 1999. Potassium bromate (KbrO3) induces renal proliferative response and damage by elaborating oxidative stress. Cancer Lett., 135: 181-188.
PubMed  |  Direct Link  |  

7:  Mack, R.B., 1988. Round up the usual suspects. Potassium bromate poisoning. North Carolina Med. J., 49: 243-245.

8:  Okolie, N.P. and E.J. Osarenren, 2003. Toxic bromate residues in Nigerian bread. Bull. Environ. Contam. Toxicol., 70: 443-446.
CrossRef  |  

9:  Watanabe, S., Y. Yoshimura and T. Fukui, 2001. Contribution of glutathione peroxidase and nitric oxide to potassium bromate-induced oxidative stress and kidney damage in mice. J. Health Sci., 47: 565-570.
CrossRef  |  

10:  Parsons, J.L. and J.K. Chipman, 2000. The role of glutathione in DNA damage by potassium bromate in vitro. Mutagenesis, 15: 311-316.
CrossRef  |  Direct Link  |  

11:  Taylor, A., 1993. Cataract: Relationships between nutrition and oxidation. J. Am. Coll. Nutr., 12: 138-146.
Direct Link  |  

12:  Taylor, A. and T. Norwell, 1997. Oxidative stress and antioxidant function in relation to risk for cataract. Adv. Pharmacol., 38: 515-536.
PubMed  |  

13:  Meister, A., 1992. Commentary on the antioxidant effects of ascorbic acid and glutathione. Biochem. Pharmacol., 44: 1903-1915.

14:  Beers, Jr. R.F. and I.W. Sizer, 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem., 195: 133-140.
PubMed  |  Direct Link  |  

15:  Misra, H.P. and I. Fridovich, 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem., 247: 3170-3175.
PubMed  |  Direct Link  |  

16:  Hesketh, J.E., J.B. Loudon, H.W. Reading and A.I.M. Glen, 1978. The effect of lithium treatment on erythrocyte membrane ATPase activities and erythrocyte ion content. Br. J. Clin. Pharmacol., 5: 323-329.
Direct Link  |  

17:  Fiske, C.H. and Y. Subbarow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem., 66: 375-400.
Direct Link  |  

18:  Gutteridge, J.M.C. and S. Wilkins, 1980. Copper-dependent hydroxyl radical damage to ascorbic acid: Formation of a thiobarbituric acid-reactive product. FEBS Lett., 137: 327-330.
PubMed  |  

19:  Roe, J.H. and C.A. Kuether, 1943. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid. J. Biol. Chem., 147: 399-407.
Direct Link  |  

20:  Jakutowicz, K., Z.I. Tomick and L. Leokadia, 1977. Deterination of total plasma tocopherol in the presence of carotenes. Pol. Arch. Weter, 20: 45-47.

21:  Kurokawa, Y., A. Maekawa, M. Takahashi and Y. Hayashi, 1990. Toxicity and carcinogenicity of potassium bromate-A new renal carcinogen. Environ. Health Perspect., 87: 309-335.
Direct Link  |  

22:  Takamura, H., Y. Kurokawa, Y. Matsushima, T. Imazawa, H. Onodera and Y. Hayashi, 1985. Long-term oral administration of potassium bromate in male Syrian hamsters. Sci. Rep. Res. Inst. Tokohu Univ. Ser. C, 32: 43-46.
PubMed  |  

23:  Nakajima, M., M. Kitazawa, K. Oba, V. Kitagawa and Y. Toyoda, 1989. Effect of route of administration in the micronucleus test with potassium bromate. Mutat. Res., 223: 399-402.
CrossRef  |  

24:  Nakano, K., S. Okada, S. Toyokuni and O. Midorikawa, 1989. Renal changes induced by chronic oral administration of potassium bromate or ferric nitrilotriacetate in Wistar rats, Naikan Hokan. Jap. Arch. Int. Med., 36: 41-47.

25:  DeAgelo, A.B., M.H. George, S.R. Kitbu, T.M. Moore and D.C. Wolf, 1998. Carcinogenicity of potassium bromate administered in the drinking water to male BLC3F1, mice and F344/N rats. Toxicol. Pathol., 26: 587-594.
CrossRef  |  PubMed  |  

26:  Murata, M., Y. Bansho, S. Inoue, K. Ito, S. Ohnish, K. Midorikawa and S. Kawanishi, 2001. Requirement of glutathione and cysteine in guanine-specific oxidation of DNA by carcinogenic potassium bromate. Chem. Res. Toxicity, 14: 678-685.
CrossRef  |  

27:  Imai, K., T. Aimoto, M. Sato and R. Kimura, 1991. Antioxidative effect of protoporphyrin on lipid peroxidation in tissue homogenates of intravenously administered rats. J. Pharmacobiodyn., 14: 20-24.
PubMed  |  

28:  Ballmaier, D. and B. Epe, 1995. Oxidative DNA damage induced by potassium bromate under cell free conditions and in mammalian cells. Carcinogenesis, 16: 335-342.
CrossRef  |  

29:  Nakae, D., T. Umemura and V. Kurokawa, 2002. Oxygen species-induced stress. Asian Pac. J. Cancer Prev., 3: 313-318.

30:  Jacques, P.F., L.T. Chy-Lack, R.B. McGandy and S.C. Hartz, 1988. Antioxidant status in persons with and without senile cataract. Arch. Ophthalmol., 106: 337-340.
PubMed  |  

31:  Baghieri, S. and M.H. Garner, 1992. Na+-K+-ATPase and phospholipid degradation in bovine and human lenses. Curr. Eye Res., 11: 459-467.

32:  Brody, T.M., T. Akera, S.F. Bashin, R. Gubitz and C.Y. Lee, 1974. Interactions of Na+-K+-ATPase with chlorpromazine free radical and related compounds. Ann. N. Y. Acad. Sci., 242: 527-530.

33:  Ratburn, W.B., 1980. Biochemistry of the lens and cataractogenesis. Curr. Concepts Small Anim. Pract., 10: 261-263.

34:  Housley, M.D. and K.K. Stanley, 1990. Dynamics of Biological Membranes Influence on Synthesis and Function. John Wiley and Sons, New York, pp: 92-151

35:  JECFA., 1989. FAO/WHO Joint Expert Committee on Food Additives Series. Cambridge University Press, Cambridge

36:  Wolf, G.W. and L. Kaiser, 1996. Final report on sodium bromate: Short-term reproductive and developmental toxicity study when administered to Sprague Dawley rats in the drinking water. National Toxicology Programme, NTP/NIEHS No 1-ES-15323.

37:  Whikehart, D.R., 2004. Biochemistry of the Eye. 2nd Edn., Port Melbourne, Butterworth-Heinemann, Oxford

38:  Young, L.G. and R.C. Augusteyn, 1993. Smoking and cataracts. 21st Annual Report of NVRI, pp: 20.

39:  Aruoma, O.I., 1994. Experimental Tools in Free Radical Biochemistry. Harwood Academic Publishers, London, pp: 233-269

40:  Clayton, R.M., D. Cuthberts and J. Seth, 1984. Epidemiological and Other Studies in the Assessment of Factors Contributing to Cataractogenesis. In: Human Cataract Formation, Nuget, W. (Ed.). Pitman, London, pp: 25-47

41:  Ishidate, Jr., M., T. Sofuni, K. Yoshikawa, M. Hayashi, M. Nohmi, T. Sawada and A. Matsuoka, 1984. Primary mutagenicity screening of food additives currently used in Japan. Food Chem. Toxicol., 22: 623-636.

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