Calcium absorption from apple and orange juice fortified with calcium citrate malate (CCM).

OBJECTIVE: Determine calcium (Ca) absorption from Ca fortified orange and apple juice. METHODS: Absorbability was assessed by measuring 45Ca absorption in healthy women (mean age 57 years, n = 57/group) and whole body 47Ca retention in adult female beagle dogs (n = 6/group) and young adult male rats (n = 6/group). Women received 6.24 mmol (250 mg) Ca as calcium citrate malate fortified orange juice (CCM-OJ) or apple juice (CCM-AJ). Dogs received 3.12 mmol (125 mg) Ca as CCM-OJ or CCM-AJ. Rats were administered 0.15 mmol (6 mg) Ca as either milk, CCM-OJ, or CCM-AJ. Additional 47Ca whole body retention experiments in rats measured the effects of differences in the carbohydrate and organic acid contents of the juices on Ca absorption. RESULTS: Mean +/- SEM percent Ca fractional absorption was greater (p < 0.003) in women who consumed CCM-AJ (42 +/- 2%) than those who consumed CCM-OJ (36 +/- 1%). Ca retention in dogs was 15 +/- 1% for CCM-OJ and 29 +/- 2% for CCM-AJ (p < 0.001). Ca retention was significantly different (p < 0.05) in rats administered milk (42 +/- 2%), CCM-OJ (52 +/- 2%), or CCM-AJ (61 +/- 2%). By manipulating the carbohydrate and organic acid concentrations of test solutions to mimic the composition of Ca fortified juices, we found that the greater fructose and lower organic acid content of apple juice accounted for its greater Ca absorbability. CONCLUSIONS: CCM fortified versions of orange and apple juice have high Ca absorbability and are potentially important vehicles for increasing dietary Ca intake. The greater Ca absorption from CCM-AJ compared with CCM-OJ is accounted for by differences in the carbohydrate and organic acid content of the juices. These data suggest that by modifying common beverage ingredients, products with even greater Ca absorbability could be formulated.

Effect of age, calcium source, and radiolabeling method on whole body 47Ca retention in the rat.

In a longitudinal study we determined the effect of animal age as well as Ca source and radiolabeling method on Ca bioavailability by measuring whole body 47Ca retention (WBR) in male Sprague-Dawley rats. The WBR assay was performed without surgery or anesthesia, and the same groups of animals were studied at 8, 16, 20, and 32 wk of age. Rats were administered a 6-mg radiolabeled oral dose of Ca as Ca citrate malate (CCM) or intrinsically or extrinsically labeled CaCO3 or hydroxyapatite (HAP). Fractional Ca retention was measured from the 72-h postdose WBR divided by WBR at time 0. WBR was significantly affected by Ca source with CCM > CaCO3 > HAP at all ages (P < 0.001). The rank order and relative bioavailabilities of these Ca salts in the rat model agreed well with literature values for Ca absorption in adult humans. Although percent WBR decreased significantly with advancing age (P < 0.001), the mean rate of decline (-3.4%/wk) was not affected by Ca source. Extrinsic radiolabeling overestimated (approximately 20%) Ca bioavailability when the rats were young. However, the magnitude of this effect diminished with advancing animal age and was not significant across all ages (repeated measures analysis of variance P = 0.10).

Intraduodenal delivery of intrinsically and extrinsically labelled CaCO3 in the rat: effect of solubilization on calcium bioavailability.

The dissolution of CaCO3 before intraduodenal administration was found to be an important factor determining calcium (Ca) bioavailability. Extrinsically and intrinsically labelled 47CaCO3 preparations were sequentially dissolved by serial additions of HCl. Aliquots of these preparations were collected before (no HCl added) and during the solubilization process and administered intraduodenally to rats. Whole body 47Ca retention 72 h post-dose was used as a measure of Ca bioavailability. Although dissolution of CaCO3 significantly increased Ca bioavailability (P < 0.001), Ca from both intrinsically and extrinsically labelled CaCO3 was absorbed and retained to some extent without prior acid dissolution. Due to a disproportionately high concentration of 47Ca on the particle surface, extrinsically labelled 47CaCO3 overestimated bioavailability when unsolubilized or partially solubilized CaCO3 preparations were used (P < 0.05). These data indicate that dissolution is a determining factor for Ca bioavailability from CaCO3. Incomplete dissolution will significantly limit but not completely prevent Ca bioavailability. The disintegration and dissolution characteristics of commercial CaCO3 preparations, which vary widely, may produce important differences in Ca absorption.

Assessment of the subchronic oral toxicity of d-limonene in dogs.

Several hydrocarbons, including d-limonene, have been shown to produce a male-rat-specific nephrotoxicity that is manifested acutely as exacerbation of hyaline droplet formation. In a study to assess the presence or absence of this response in a non-rodent species, the dog was selected as a relevant model because of an earlier report suggesting that d-limonene may be nephrotoxic in this species. Five male and five female adult beagle dogs per treatment group were gavaged twice daily over a 6-month period with tap-water (control) or d-limonene at 0.12 or 1.2 ml/kg body weight/day (100 or 1000 mg/kg body weight/day). The highest daily dose was determined in a pilot study to be close to the maximum tolerated dose for emesis (ED50 1.6 ml/kg body weight). The test compound was administered in divided doses to minimize the incidence of emesis. Feed consumption and body weight were unaffected by treatment. Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys, evaluated by both haematoxylin and eosin and Mallory-Heidenhain staining, that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation nor of any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene. Thus, dogs are refractory to the hyaline droplet nephropathy observed in male rats, thereby providing additional evidence that the male rat kidney is uniquely sensitive to hydrocarbons like d-limonene, and that this specific male rat nephropathic response may be inappropriate for interspecies extrapolation and human risk assessment.

Calcium bioavailability and iron-calcium interaction in orange juice.

We have determined the effects of orange juice on calcium bioavailability from CCM (a combination of CaCO3, citric acid, malic acid, 5:1:1, mol/mol/mol) and iron-calcium interaction by using whole body isotope retention techniques in rats. The mean calcium retention values from CCM were 42.8% from orange juice and 33.0% from water, a control. Orange juice significantly (p less than 0.05) improved calcium bioavailability. This enhancement of calcium absorption is independent of orange juice's pH and citric acid. Iron absorption from orange juice with CCM (36.7%) was also significantly higher than that from control (water) plus CCM (12.3%). Ascorbic acid at levels naturally present in orange juice failed to improve iron retention (12.3% vs 12.5%) from water plus CCM. In contrast, citric acid (at orange juice level) significantly (p less than 0.05) promoted iron absorption in the presence of CCM (8.0% vs 23.7%). The benefit of citric acid on iron-calcium interaction is enhanced by ascorbic acid. In the presence of both citric acid and ascorbic acid, at orange juice levels, iron absorption from water plus CCM (37.6%) was comparable to that from water without CCM (34.5%). These results show orange juice can deliver bioavailable calcium from CCM with minimal inhibition of iron absorption. Citric acid and ascorbic acid are likely the major orange juice components that contribute to the alleviation of iron absorption inhibition by CCM.

Effects of variation of necropsy time and fasting on liver weights and liver components in rats.

In rats, percent liver weight loss is greater than percent body weight loss within the 8 A.M.-4 P.M. period of the working day. The liver weight loss is principally the result of decreased water content, either carbohydrate (glycogen) bound water in rats with access to feed, or protein bound water in rats fasted overnight. During this period, percent kidney weight loss is approximately equal to percent body weight loss. To optimize the sensitivity of kidney and liver weight evaluation, it is recommended that rats be fasted overnight and that relative liver and kidney weights be expressed based on body weights taken immediately prior to necropsy. Since these procedures will not entirely eliminate necropsy time-related organ weight differences, the animal necropsy sequence must be randomized to distribute the remaining differences across all treatment groups.

Comparison of short-term renal effects due to oral administration of decalin or d-limonene in young adult male Fischer-344 rats.

Groups of young adult male Fischer-344 rats given the vehicle (corn oil) or either decalin or d-limonene at dose levels of 75, 150 or 300 mg/kg body weight by a single daily gavage on 5 days/wk were killed on study days 6 or 27, approximately 24 hr after the fifth or 20th dose, to determine whether the specific time- and dose-related triad of renal alterations characterizing decalin-associated nephrotoxicity in the adult male rat also occurs in response to d-limonene. Dose-related hyaline droplet formation associated with renal accumulation of a specific protein alpha 2u-globulin) is considered the primary response in the morphogenesis of decalin-induced nephrotoxicity in the male rat and was present to a maximal degree in all decalin- and d-limonene-treated groups by day 6. Alterations considered to be sequelae of the hyaline droplet response, including granular casts in the outer zone of the medulla and multiple cortical changes collectively classified as chronic nephrosis, were present in the kidneys of both decalin- and d-limonene-treated rats killed on day 27. These findings demonstrate a uniformity of primary and secondary renal responses to the two chemicals, strongly suggesting that the morphogenesis of d-limonene-associated nephrotoxicity in the adult male rat is consistent with that of decalin. The response of the male rat kidney to decalin treatment has been shown to be uniquely different, by virtue of anatomical, physiological and biochemical peculiarities involving the proximal convoluted tubule, from that in female rats and higher mammalian species.

Review of kidney sections from a subchronic d-limonene oral dosing study conducted by the National Cancer Institute.

d-Limonene administered by oral gavage at 150-2400 mg/kg/day in a subchronic (91-day) study conducted for the National Cancer Institute induced renal alterations in male rats at all dose levels, whereas kidneys of male mice, female rats and female mice were unaffected. The renal alterations were dose responsive, and were similar to changes observed as sequelae to oral or inhalation exposure to decalin, a model compound used in a volatile hydrocarbon toxicology programme. Decalin induces a nephrotoxic response unique to the male rat, but the primary response associated with decalin exposure--hyaline droplet formation within the cytoplasm of the epithelial cells of the proximal convoluted tubule--was not recognized in the kidneys of d-limonene-exposed male rats. A possible explanation for the absence of this primary response from kidneys of the d-limonene-treated male rats could be the 4-5-day interval between administration of the final dose and the killing of the animals.

Development of a short-term model of decalin inhalation nephrotoxicity in the male rat.

Fischer 344 male rats and C57BL/6 male mice were exposed 'continuously' (22 hr/day, 7 days/wk) for 20, 28 or 35 days to a model compound, decalin, at 0, 25, 62.5 or 125 ppm. Fischer 344 female rats were exposed 'continuously' to decalin at 0 or 125 ppm for 28 days. No histopathological changes were observed in selected organs of female rats or male mice exposed to up to 125 ppm decalin for 28 or 35 days, respectively. However, kidney lesions were observed in all three test groups of male rats after 20, 28 and 35 days' exposure. The nephrotoxicity was characterized by the formation of hyaline droplets in the cytoplasm of proximal convoluted tubule epithelial cells, by the presence of granular casts at the outer zone of the medulla, and by chronic nephrosis. These changes were time and dose dependent and were identical to the renal toxicity that has been reported to occur in male rats following 90 days of continuous exposure to decalin by inhalation. No histopathological effects were observed in the heart, liver, lung or nasal turbinates of male rats. Our results indicate a sex and species specificity for the kidney toxicity. This leads to questions with regard to the appropriateness of using the male rat to assess the potential inhalation toxicity of volatile hydrocarbons. By producing nephrotoxicity in less than 90 days, decalin may now be used to examine, in a well-defined manner, the effect on nephrotoxicity of variables such as dose, exposure regimen, sex, species, and route of exposure. Data from these studies can be used to ascertain whether or not the male rat is an appropriate test animal for predicting potential human nephrotoxic responses to volatile chemicals such as perfumes and perfume raw materials.

Decalin-induced nephrotoxicity: light and electron microscopic examination of the effects of oral dosing on the development of kidney lesions in the rat.

Male and female Fischer 344 rats were given decalin by oral gavage for 5 or 12 consecutive days in order to determine whether oral dosing would result in light microscopically evident renal effects that were comparable to those that have been observed after inhalation exposure. Decalin (in corn oil vehicle) was administered at doses of 0, 0.1, 0.5, 1.0 or 2.0 g/kg body weight to male rats, and 0, 1.0, 1.5, 1.75 or 2.0 g/kg to female rats. Biopsies of the kidneys of selected control and high-dose male rats were taken for examination by electron microscopy. Sections of kidneys from all control and treated rats were examined by light microscopy. The kidneys of all male control rats contained minimal levels of hyaline droplets within the cytoplasm of proximal convoluted tubule (PCT) epithelial cells. Decalin-induced alterations in the kidneys of male rats included an exacerbation of the hyaline droplet/globule levels found in controls and the formation of granular casts in the outer zone of the renal medulla. The exacerbated formation of hyaline droplets was characterized light microscopically by a marked dose-related increase in the number and size of individual droplets/globules and ultrastructurally by a marked increase in the size range of, and the presence of crystalline inclusions in, the PCT epithelial cell phagolysosomal populations. No other ultrastructural alterations occurred that differentiated treated male rats from control males. The formation of granular casts was dose and time related, occurring in 60% of male rats given 0.5 g decalin/kg for 12 days and in 100% of those given 1.0 g decalin/kg for 12 days. Light microscopy revealed no differences between the kidneys of control and decalin-treated female rats, and no hyaline droplets or granular casts were observed in the kidneys of any female rat killed after 5 or 12 days. These results were in agreement with those of inhalation studies and provide additional evidence that the formation of hyaline droplets in response to exposure to volatile hydrocarbons may be unique to the male rat.

Morphogenesis of decalin-induced renal alterations in the male rat.

Adult male Fischer 344 rats were killed after 5, 12, 19 or 31 days' 'occupational' (6 hr/day, 5 days/wk), 'semi-continuous' (22 hr/day, 5 days/wk) or 'continuous' (22 hr/day, 7 days/wk) exposure to 125 ppm decalin vapour. Control rats were exposed to filtered air. Kidney sections were evaluated to determine the nature and time-course of development of decalin-induced lesions. The development of renal lesions was characterized by a specific sequence of light microscopically evident alterations. The extent of the alterations was dependent on time and exposure regimen. Severe exacerbation of the spontaneous protein accumulation (hyaline droplets) routinely observed in the kidneys of control male rats was present in kidneys of all decalin-exposed animals at day 5, and was considered to be the primary morphological alteration associated with decalin exposure. The following sequelae of the hyaline droplet response were observed: the variable occurrence of light microscopically evident proximal convoluted tubule (PCT) epithelial cell degeneration/necrosis, presumably a reflection of cellular injury associated with excessive protein accumulation; the occurrence of granular casts at the junction of the inner and outer bands of the outer zone of the medulla secondary to PCT epithelial cell injury; chronic nephrosis, occurring secondary to tubular obstruction by granular casts. This triad of lesions (hyaline droplet accumulation, granular cast formation and chronic nephrosis) lends specificity to the decalin response and establishes a potential mechanistic relationship with other chemicals that induce these effects.

Characterization of spontaneous and decalin-induced hyaline droplets in kidneys of adult male rats.

Studies were conducted to gain additional information about the spontaneous and decalin-exacerbated formation of hyaline droplets within the cytoplasm of proximal convoluted tubule (PCT) epithelial cells of the adult male rat. Renal cortical tissue protein patterns determined through two-dimensional gel electrophoresis revealed four species of a low-molecular-weight protein (18,000-20,000 daltons). Treatment groups differed only with respect to this protein, the relative concentrations of which paralleled the numbers of hyaline droplets in mature treated and untreated male rats. The increase in the numbers of hyaline droplets and protein accumulation were dose related. Neither this protein or hyaline droplets were detected in the renal cortical tissues of untreated or decalin-exposed adult female or immature male control rats. However this protein, and hyaline droplet formation, could be induced in the kidneys of adult, ovariectomized female rats by repeated testosterone injections. This protein was then demonstrated to be immunologically identical to alpha 2u-globulin, a protein synthesized by hepatic parenchymal cells. Alpha 2u-globulin protein has also been shown to be the major urinary component responsible for the proteinuria routinely observed in normal control adult male rats. PCT epithelial cell reabsorption and lysosomal accumulation of alpha 2u-globulin, reflected morphologically as hyaline droplets, occurs spontaneously only in the mature male rat. Decalin, a model compound, exacerbates this accumulation as a specific integral step in the pathogenesis of the nephropathy induced in male rats by volatile hydrocarbons. Hence, since men and women lack this specific PCT cell peculiarity, they would not be expected to respond to decalin exposure in a manner similar to the male rat.

Effect of N-nitroso-n-butyl-(4-hydroxybutyl)amine exposure on the changes in mineral disposition caused by trisodium nitrilotriacetate.

Male Fischer 344 rats were used to determine effect of consumption of 0.5% N-nitroso-n-butyl-(4-hydroxybutyl)amine (BNN) in the drinking-water for 2 wk on the response to 0.02, 0.2 and 2.0% trisodium nitrilotriacetate (Na3 NTA . H2O) in the diet in terms of urinary mineral excretion, bladder mass and bladder mineral concentrations. The primary objective of the study was to determine whether exposure of rats to an initiating dose of a bladder carcinogen (BBN) alters the threshold dose of Na3NTA . H2O required to alter urinary or bladder mineral concentrations or the dose-response to NTA. Such alterations are considered to be necessary precursors for changes in bladder morphology in rats fed NTA in chronic toxicity studies (Anderson, Bishop & Campbell, CRC Crit. Rev. Toxicol. 1985, 15, 1). The results demonstrated that BBN exposure caused an increase in bladder mass and bladder-tissue Zn concentration. However, BBN pretreatment did not have any effect on Na3NTA . H2O metabolism, the threshold dose of Na3NTA . H2O required to attain the necessary conditions for induction of bladder toxicity by NTA, or the dose-response relationships for NTA's effects on any parameter examined. From these data, it is concluded that it is unlikely that NTA would show a different threshold or dose-response for bladder tumour promotion than for its tumorigenicity at this site, which has been demonstrated previously (National Cancer Institute, DHEW Publication No. (NIH) 77-806, 1977).

Renal pelvic and ureteral dilatation in male rats ingesting trisodium nitrilotriacetate.

Renal pelvic and proximal ureteral dilatation has been observed in male Charles River (CD) and Fischer 344 rats ingesting comparable doses of Na3NTA X H2O. Ureteral dilatation is accompanied by epithelial surface ulceration and erosion. Taken together with previous work (Anderson, Alden & Merski, Fd Chem. Toxic. 1982, 20, 105), these findings demonstrate that the ingestion of high doses of NTA results in similar effects on the transitional epithelium throughout the urinary tract.

Comparison of fresh and fixed organ weights of rats.

Male and female rats (20 of each sex) were killed, and a broad sample of organs were excised, weighed, and immersed in 10% neutral buffered formalin. Following 72 hours fixation the organs were reweighed. Comparison of fresh and fixed organ weights revealed statistically significant organ weight changes due to fixation. Although the fixation-induced organ weight changes varied in both direction and magnitude among organs and between sexes, the changes were consistent throughout samplings of each specific organ. The results of this study therefore suggest that fixed organ weights may be a valid alternative to fresh organ weights. A significantly larger data base must be generated, however, to determine the influence of fixation prior to weighing in the presence of the various pathologic tissue alterations observed in safety evaluation studies.

Reversibility of nephrotoxicity induced in rats by nitrilotriacetate in subchronic feeding studies.

The reversibility of nitrilotriacetate (NTA)-associated nephrotoxicity was investigated by comparing renal tissues from rats fed nephrotoxic levels of NTA for 7 wk with those from rats allowed 5 wk of recovery after the 7-wk exposure. In addition the toxicity of 2% Na3NTA X H2O in the diet (73 mumol/g diet) was compared with that of 1.5% H3NTA (79 mumol/g diet). The two forms of NTA induced comparable renal tubular cell toxicity which was characterized by proximal convoluted cell vacuolation and hyperplasia. These effects were noted in all of the exposed animals although the extent of damage varied. This specific renal tubular cell toxicity was completely reversed during the 5-wk recovery period. Renal pelvic transitional cell toxicity was induced primarily by Na3NTA X H2O. Renal pelvic toxicity was characterized by hydronephrosis, and erosion, ulceration and hyperplasia of the transitional epithelium. All forms of renal toxicity except that accompanying hydronephrosis were reversed when Na3NTA X H2O feeding was discontinued.

Reversibility of renal cortical lesions induced in rats by high doses of nitrilotriacetate in chronic feeding studies.

The effects in the renal cortex of the male rat of continuous administration of a high dose of nitrilotriacetate (NTA) for 24 months were compared with those of the administration of a similar dose for 18 months followed by a 6-month recovery period on the control diet. The results suggest that discontinuation of treatment interrupts the sequence of events leading to tumour formation. This study indicates that all stages in the proposed pathogenesis of renal tubular tumour formation by NTA, up to the occurrence of adenomatous hyperplasia and neoplasia, are reversible. Apparently, tumour development is dependent on the continuous administration of high doses of NTA in the presence of toxic injury to the majority of nephrons.

The pathogenesis of renal cortical tumours in rats fed 2% trisodium nitrilotriacetate monohydrate.

To help assess the relationship between the renal toxicity and tumorigenicity associated with the administration of high doses of nitrilotriacetate (NTA) we have reviewed slides of sections from the kidneys of rats used in the National Cancer Institute bioassay of NTA. Trisodium NTA fed to Fischer 344 rats at 2% in the diet for 2 yr exerts a persistent toxic effect on the renal cortex which is manifested morphologically as vacuolation of proximal convoluted tubular epithelium and exacerbation of age-related nephrosis. Additionally, two pathogenic pathways leading to tumour formation in NTA-treated rats are suggested. A specific pathway initiated by vacuolation of proximal convoluted tubular epithelium leads to hyperplasia; reasons are advanced for the view that hyperplasia progresses to neoplasia. A possible concomitant pathway, which we suggest is nonspecific, is associated with regenerative proliferation in kidneys affected by severe age-related nephrosis. These data support the concept that there is a causal relationship between NTA-associated tubular toxicity and tumorigenicity. Doses of NTA that do not induce toxicity do not induce tubular tumours as demonstrated in this and in two other major long-term studies. Hence studies to investigate the pathogenesis of the toxic response provide an insight that suggests the basis for the development of NTA-associated tumours.

Short-term effects of dietary nitrilotriacetic acid in the male Charles River rat kidney.

Trisodium nitrilotriacetate monohydrate was fed to male weanling Charles River rats for 28 days. Clinical evidence of urinary tract toxicity included hydronephrosis and nephromegaly. Microscopically, severe renal injury was recognized specifically as convoluted tubular cytoplasmic vacuolation and pelvic epithelial erosion. Hyperplasis occurred as a sequela to these specific cytotoxic alterations.