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1,
*
Roche Vitamins Incorporated, Nutley, NJ 07110;
Department of Preventive Medicine and Community Health, UMDNJ-New Jersey Medical School, Newark, NJ 07103;
**
Battelle, Incorporated, Columbus, OH 43201; and
MPI Research LLC, Mattawan, MI 49071
1To whom correspondence should be addressed. E-mail: bogden{at}umdnj.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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250g; n = 20/group) consumed diets with adequate (40 µg/g), deficient (<2 µg/g) or fortified (100 µg/g) zinc concentrations ad libitum for 28 d. Two groups fed either Zn-adequate or Zn-fortified diets also were given 100 mg/(kg · d) of FB in diet, and 2 groups were pair-fed controls. Histopathology or metal analyses were performed on tissues from 10 rats/group. FB caused EH of the nonglandular stomach but not of other tissues. Of the metals evaluated, only copper concentrations were significantly reduced in all tissues examined except kidney. A broad range of kidney copper concentrations was found; these concentrations were associated with plasma copper and proteinaceous deposits within tubules. In rats, FB substantially and selectively depletes Cu in vivo, suggesting that drugs with structures that permit metal chelation should be evaluated for their potential to alter trace metal nutriture.
KEY WORDS: • copper • Frenolicin-B • antibiotics • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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.,5..,11b..)–3,3a,5,11b-tetrahydro-7-hydroxy-5-propyl-2H-furo[3,2-b]naphtho[2,3-d]pyran-2,6,11-trione, CAS#68930–68-7] is a member of the polyketide family of antibiotics that has been the subject of considerable investigation for their efficacy as growth enhancers for poultry and other farm animals, thus having the potential to be used in very substantial quantities. They may also be suitable for human use.
FB also has been reported to chelate divalent cations in vitro (J. Zink, UCLA, personal communication). In preliminary toxicology studies, rodents treated with dietary FB developed anorexia and epithelial hyperplasia (EH) of the esophagus and nonglandular stomach after 30 d of treatment (D. Kossor, unpublished observations). Although EH of the nonglandular stomach in rats is a relatively common toxicological finding that is associated with the oral administration of irritant compounds (1
–5
), esophageal EH is not as common. Previous studies to evaluate this lesion utilized a well-known rodent laboratory model of esophageal EH, which results from feeding a Zn-deficient diet (6
,7
). We therefore hypothesized that FB may induce EH in rats by chelating Zn in the diet and/or in vivo, leading to clinical Zn deficiency.
The objectives of this study were as follows: 1) to determine whether FB causes EH in oral or upper gastrointestinal epithelial tissues in rats; 2) to determine whether FB causes zinc deficiency in rats fed two different levels of dietary zinc; and 3) to determine whether FB causes deficiency of one or more of the essential metals calcium, copper, iron and magnesium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For these studies, 175 Sprague-Dawley (Crl:CD VAF/Plus) rats, 4 wk old, were obtained from Charles River Laboratories (Portage, MI). Rats were housed individually in stainless steel wire-mesh type cages and were acclimated for 4 wk before study initiation. Water was continually available. Rats were maintained on a 12-h light:dark cycle, and temperature (22–23°C) and humidity (45–68%) were monitored and controlled. Rats were handled in accordance with the NIH guidelines for the care and handling of laboratory animals (8
).
Chemicals.
Frenolicin-B (95% wt/wt) was obtained from Roche Vitamins (Nutley, NJ); other chemicals were obtained from commercial sources.
Diets.
Diets containing deficient (<2 µg/g), adequate (40 µg/g) and fortified (100 µg/g) Zn concentrations were prepared by Dyets (Bethlehem, PA). This was done by first preparing an egg white–based diet (Zn-deficient; AIN-93G) to which appropriate amounts of zinc carbonate were added to achieve the desired final Zn concentrations (9
). Samples of each diet were collected and analyzed by atomic absorption spectroscopy to verify the appropriate Zn concentrations. To formulate diets, FB was dissolved in acetone and incorporated into the diet using geometric dilutions. Adequate mixing time was provided to permit the acetone to volatilize during preparation. After formulation with FB, diets were analyzed by HPLC for the target FB concentrations, and were found to be satisfactory with respect to concentrations, homogeneity and stability. Based on rat body weights and diet consumption, the formulation was changed weekly to achieve a dose of 100 mg/(kg · d), one that provides a 10- to 20-fold safety factor compared with lower doses typically used in commercial feeds.
The target diet concentrations for zinc were <2, 40 and 100 µg/g; mean values by analysis were 1.36, 39.8, and 96.4 µg/g, respectively, based on three replicates. Target diet concentrations for Cu, Fe, Mg, and Cu were 6.0, 40, 700 and 5000 µg/g, respectively; the corresponding mean values by analysis (9 replicates) were 5.89, 42.5, 712, and 5090 µg/g, respectively.
Study design.
Rats were randomly assigned to seven treatment groups (n = 20/group). Groups 1, 4 and 7 consumed ad libitum diets that were Zn-adequate (40 µg/g Zn), Zn-fortified (100 µg/g Zn) (hereafter referred to as "ad libitum controls") or Zn-deficient (<2 µg/g Zn), respectively. Rats in groups 3 and 6 were given diets that contained FB [100 mg/(kg · d)] in either a Zn-adequate or Zn-fortified diet, respectively. Rats in groups 2 and 5 were given the Zn-adequate or Zn-fortified diet, respectively, and were pair-fed to the corresponding FB-treated group. Treatments were administered to all groups for 28 d. Pair feeding was done on a daily basis; each rat was given a quantity of diet equal to the mean amount consumed by the respective FB-treated group during the preceding day.
Necropsy/Histopathology.
Rats were killed by carbon dioxide inhalation after blood samples were collected via cardiac puncture into heparinized tubes. Plasma was isolated by centrifugation for 10 min at 1150 x g. Selected organs from 10 rats per group (brain, submaxillary salivary gland, tongue, esophagus, nonglandular stomach, kidney and femur) were collected, trimmed of connecting tissue, weighed and fixed in formalin. Tissue sections (5 µm) were prepared and stained with hematoxylin and eosin for light microscopic evaluation. Organs from the remaining 10 rats per group were rinsed briefly with distilled water, air-dried and stored in prerinsed polyethylene containers before trace metal analysis.
Metal analyses.
Organs for trace element analyses were placed in individual polyethylene bags (prerinsed with deionized water before use) and stored at -70°C. During tissue collection, precautions were taken to avoid any potential trace element contamination. Only stainless steel instruments came into contact with the tissues; only plastic containers were used for the tissues; all containers and equipment were rinsed thoroughly in deionized water and allowed to dry before use; and the tissues did not come into contact with any contaminating surfaces, such as blotter paper or toweling. Organ concentrations of calcium, copper, iron, magnesium and zinc were determined by previously described techniques using flame atomic absorption spectrophotometry (10
). National Institutes of Standards and Technology bovine liver (SRM 1577b, Gaithersburg, MD) was used as a quality control sample for all analyses. Assays of this sample in our laboratory gave results within 5% of certified values. Calculations of concentrations were based on wet tissue weight.
Statistical analyses.
Data reduction and analyses were performed using dBase III+ (Ashton-Tate, Torrance, CA) and the Statistical Analysis System (SAS Institute, Cary, NC). ANOVA was used to evaluate the effects of FB on organ metal concentrations and contents. If ANOVA indicated that there were significant (P < 0.05) differences among the seven treatment groups for a specific measurement, then pair-wise comparisons were made by Duncan’s multiple range test at = 0.05 (11
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Increased relative kidney weight in FB-treated rats was associated with histopathological findings in the kidney that included dilatation of the convoluted tubules accompanied by the deposition of proteinaceous material in the tubular lumen. These changes were observed in all rats treated with FB, and there were no differences in the morphology, severity or incidence of lesions between the groups that received FB in either Zn-adequate or Zn-fortified diets.
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Table 2
contains concentrations of zinc, copper, and iron in blood plasma and the seven organs studied. Calcium and magnesium concentrations and data for group 7 (Zn-deficient diet) are not included in Table 2
but are summarized below. The diets containing FB caused moderate-to-substantial decreases in copper concentrations in plasma and all organs except kidney for which an atypically broad range of concentrations was found (Table 2)
. There were significant (P < 0.01) associations between plasma and kidney copper of FB-treated rats fed either the zinc-adequate (r = 0.976) or zinc-fortified diets (r = 0.895).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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90 µg/dL (14 µmol/L) after 4 wk of FB ingestion is comparable to decreases reported by some other investigators in rats fed Cu-deficient diets. For example, Wapnir and Devas (12
) fed a Cu-deficient diet (0.6 mg/kg) to rats for 3 wk; plasma copper concentrations decreased by 83 µg/dL (13 µmol/L). Other investigators have reported larger decreases in plasma Cu to concentrations of 5 µg/dL (1 µmol/L) in rats fed Cu-deficient diets for 5–6 wk (13
,14
).
Small but significant decreases in Cu concentrations in the brain of FB-treated rats (Zn-adequate and Zn-fortified diets) were observed, which were 92 and 88% of pair-fed controls, respectively. Studies using Cu isotopes have shown that Cu turnover in the brain in rats is restricted to <2% over a period of 56 d (15
,16
), presumably due to the presence of a relatively impermeable blood-brain barrier (17
). Thus, the present study suggests that FB treatment facilitates the net efflux of Cu from the brain. Of the organs evaluated, the nonglandular stomach had the greatest FB-mediated loss of Cu, for which values were 66 and 59% of pair-fed controls (Zn-adequate and Zn-fortified diets, respectively). The most striking histopathological changes in FB-treated rats also were observed in the nonglandular stomach, and were characterized by moderate EH, moderate hyperkeratosis and trace to mild inflammation in all examined rats fed FB. There were no differences in the morphology, severity or incidence of lesions between the two groups that received FB in either the Zn-adequate or Zn-fortified diet. Consequently, a potential role for Cu deficiency in the mechanism for FB-induced EH of the nonglandular stomach merits investigation.
Although the kidney Cu concentrations did not differ between control and FB-treated rats, a much broader range of Cu concentrations existed in FB-treated rats, suggesting that FB treatment perturbed kidney Cu homeostasis. The wide range of kidney Cu concentrations in FB-treated rats was linearly correlated with the plasma Cu concentration. Although the major excretory route for plasma Cu is via bile (18
–20
), studies have shown that renal Cu elimination is increased after treatment with chelating compounds (21
–25
). Moreover, FB treatment of rats fed both Zn-adequate and Zn-fortified diets also was associated with significant increases in relative kidney weight, compared with pair-fed controls. Together, these data suggest that rather than, or in addition to interfering with dietary Cu absorption, FB appears to promote renal Cu excretion, and saturation of excretory pathways in the kidney may explain the observed Cu deposition in the kidneys of some rats. Further research will be required to elucidate the exact mechanism for the FB-induced accumulation of Cu in the kidney of male rats.
Many drugs (10
,26
) have chemical structures that permit chelation of Zn, Cu and other trace metals. Although the potential for drugs to chelate trace elements has raised concerns concerning their bioavailability, it is possible that many side effects of drugs also may be due in part to trace element chelation and drug-induced trace element deficiencies.
In conclusion, data from the present study suggest that oral administration of FB to male rats substantially decreased tissue and plasma Cu concentrations but not those of the other four metals studied, suggesting that FB is a potent and selective chelator of Cu in vivo. Screening of candidate drugs for potential adverse effects on trace element nutriture before FDA approval for use in humans or animals is suggested.
Manuscript received April 2, 2001. Initial review completed May 9, 2001. Revision accepted September 10, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Wapnir, R. A. & Devas, G. (1995) Copper deficiency: interaction with high-fructose and high-fat diets in rats. Am. J. Clin. Nutr. 61:105-110.
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