* Safety Testing Laboratory and
Research Division, Food and Drug Safety Center, Hatano Research Institute, 729-5, Ochiai, Hadano, Kanagawa 257-8523, Japan
Received August 6, 1999; accepted March 6, 2000
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ABSTRACT |
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INTRODUCTION |
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Diet is also an essential factor influencing biologic processes such as growth, reproduction, survival, and responses to xenobiotics (Clarke et al., 1977; Gaillard et al., 1977
; Garland et al., 1989
; National Research Council, 1995; Rao, 1996
). Many investigators have reported that dietary factors altered the toxicity of various chemicals (Carrera et al., 1979
; Gans, 1982
; Hathcock, 1976
; Meyer et al., 1978
; Shively et al., 1986
). In addition to the dosing vehicle, the diet that is fed to the animals is an important factor in toxicological studies.
Recently, we had a confusing experience in a reproductive and developmental toxicity study, using corn oil at a dose rate of 10 ml/kg/day as the vehicle, in which several female rats from both the treated and the vehicle control groups did not care for their pups. From the results of microscopic observations of major organs in the dams, it was noted that almost all of the dams that had neglected their pups had severe lesions in the kidney, i.e., necrosis of the proximal tubular epithelium. This severe damage of the kidney, however, was not observed in male rats dosed with the same volume of corn oil and fed the same diet. Consequently, the diet type was changed and the dosing volume of corn oil administered was reduced, and the incidence and severity of the kidney injuries in dams were decreased. Although it is well known that the physiology of the kidney changes during pregnancy and the lactation period (Kincaid-Smith, 1985; Lindheimer and Katz, 1985
; Stock and Metcalfe; 1994), how corn oil and/or diet influenced the kidney of pregnant and lactating rats in these studies is unknown.
In this study, we evaluated whether corn oil gavage can affect the pregnant and lactational states in rats, given two different diets. In addition, histopathological examination of the kidney was performed to clarify whether the combination of corn oil and different diets may influence the maternal kidney during gestation and lactation.
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MATERIALS AND METHODS |
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Diets.
One-half of the rats of each sex were fed the CA-1 diet, and the other half were fed the CE-2 diets, both of which were closed-formula commercial pellets for rodents (CLEA Japan, Inc., Japan). The CA-1 had been developed for reproduction in inbred animals, and the CE-2 is widely used for reproduction, growth, and maintenance of rodents. Both diets have been used as the standard diet in the reproductive and developmental studies in our laboratory. The nutrient composition of the CA-1 and the CE-2 diets is shown in Table 1. Details of vitamin and mineral components of these diets are given in Table 2
. These values for both diets are at adequate levels for growth, maintenance, and reproduction, as recommended by the National Research Council (1995). The principal difference in the two diets is the source of protein; the protein in CA-1 is mainly animal protein (white fish meal and animal liver), whereas the protein in CE-2 is mainly plant protein (soybean meal), according to the catalog of CLEA Japan, Inc. These two diets were not shown to be contaminated with bacterial and chemical contaminants.
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For 2 weeks following the treatment period, each female was housed with one male within the same diet group until evidence of mating (presence of copulatory plug or sperm in a vaginal smear) was observed. In case of no evidence of mating for 2 weeks of the mating period, the females were sacrificed (see below). After mating had been noted, the females were caged individually. The day of mating was regarded as day 0 of gestation. Pregnant females were weighed and their food consumption recorded on days 0, 7, 14, and 20 of gestation. All pregnant females were allowed to deliver naturally, and day 0 of lactation was defined as the day on which all pups in a litter had been delivered. The confirmation of delivery was performed from 9 A.M. to 11 A.M. because of treatment time (from 9 A.M. to 12 P.M.). Dams were weighed and food consumption was recorded on days 0 and 4 of lactation. Litters were evaluated for the number of pups (live and dead), sexed, and weighed individually on days 0 and 4 of lactation. Visible physical abnormalities or abnormal demeanor of the pups was recorded during the lactation period.
Autopsy.
All adult females were exsanguinated under pentobarbital anesthesia on day 4 of lactation in dams, on day 25 of gestation in the case of mated females that did not deliver, and on the next day after the mating period in females that failed to copulate. Animals observed to be moribund during the course of the study were euthanized and autopsied. The uteri and ovaries were excised, and the numbers of corpora lutea and implantation sites were recorded. The kidneys, liver, and thymus of all females were weighed and preserved in 10% neutral buffered formalin. All pups were euthanized by ether and necropsied on day 4 of lactation. Pups found dead during the lactation period were also examined macroscopically.
Histopathologic examination of the kidney.
The kidneys of all adult females were examined microscopically. The fixed kidneys were embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin. The lesions of the kidneys were graded. The grading criteria were as follows: grade 0, no lesions; grade 1, very slight; grade 2, slight; grade 3, moderate; grade 4, severe.
Statistical evaluation.
Data obtained herein were analyzed, where appropriate, to detect the statistical significance of difference at p < 0.05 and p < 0.01. Two-way (diet and corn oil volume) analysis of variance (ANOVA) was used to analyze results for the body weight, food consumption, and reproductive indices. When necessary, an LSD post hoc analysis was used to evaluate more detail. If an interaction was indicated, mean comparisons were done within each diet group by one-way ANOVA. The frequency of each lesion in the kidneys was analyzed with Fisher's exact probability test for incidence and Mann-Whitney U test for trend of grade. Correlation between the grade of kidney lesions and reproductive index was performed using Spearman's rank correlation.
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RESULTS |
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Food Consumption and Body Weight Gain
Figure 1 shows the food consumption of females in each group. Two-way ANOVA indicated significant diet and corn oil volume effects in each period of the study. Diet and corn oil volume interaction effects were also detected during the lactational period. Post hoc analysis indicated the mean food consumption in both 10 ml/kg corn oil groups were significantly lower (p < 0.01) than the relevant control group except for the lactational period. On days 04 of lactation, corn oil volume effects on mean food consumption were statistically significant in each diet group (one-way ANOVA, p < 0.01). No appreciable changes in food consumption were noted in the 2 ml/kg corn oil groups of both diet groups.
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External and visceral examination of pups that died during lactation or were killed on day 4 of lactation showed no abnormality, except for emaciation in dead pups from dams in the 10 ml/kg corn oil groups fed the CA-1 diet.
Histopathology
Histopathologic findings (Table 4, Fig. 3
) of the kidneys in the 10 ml/kg corn oil groups fed the CA-1 diet showed severe epithelial necrosis and fatty degeneration of the proximal tubule. Basophilic tubules suggesting epithelial regeneration were observed as well. In comparison with the control group fed the same diet, both the incidence and the severity of necrosis (p < 0.05, p < 0.05), fatty degeneration (p < 0.05, p < 0.01), and basophilic epithelium of proximal tubules (p < 0.05, p < 0.05) were significantly increased in the 10 ml/kg corn oil groups fed the CA-1 diet. Females with severe fatty degeneration of proximal tubules tended to have both necrosis and basophilic tubules. The incidence, but not severity, of fatty change, which may be attributed to corn oil treatment, was significantly increased (p < 0.05) in the 2 ml/kg group fed the CA-1 diet and the 10 ml/kg group fed the CE-2 diet.
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DISCUSSION |
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According to a study on acute oral toxicity of corn oil, capillary venous congestion, necrosis, and many droplets at the proximal and distal tubules were observed in the kidneys of rats that died (Boyd et al., 1969). These renal lesions observed in rats treated with huge amounts of corn oil resembled our results. In addition to histopathologic alteration of the kidney by corn oil, Bachmann and Weber (1990) reported altered renal function. In female rats given a corn oil-enriched diet for 4 weeks, energy metabolism in the kidney changed, and ATP content in the kidney decreased markedly. Therefore, the kidney lesions observed in this study may be induced by corn oil administration, and it seems that the CA-1 diet enhances corn oil toxicity.
Food consumption of rats given 10 ml/kg corn oil in both diet groups was significantly lower than the controls throughout the treatment period, and the rats consumed approximately 70% of the corresponding control value. Moreover, less CA-1 diet was consumed than CE-2 diet by comparison between the groups treated with same dose of corn oil. It can be concluded that rats given 10 ml/kg corn oil and fed the CA-1 diet had the worst nutritional status among all experimental groups.
There are many reports about dietary fiber-altered toxicity of various chemicals (Chadwick et al., 1978; Kritchevsky, 1977
; Wise and Gilburt, 1980
). Although dietary fiber is not a nutrient because it passes unchanged though the intestine, dietary fiber may have influences on metabolism of xenobiotics (Johansson, 1970
; Morgan et al., 1974
). The CA-1 diet contains less fiber than the CE-2 diet, and food consumption of CA-1 was reduced compared with that of CE-2. This fiber deficiency may be one of the reasons for enhancement of corn oil toxicity in rats treated with 10 ml/kg corn oil and fed the CA-1 diet.
In the kidneys of rats treated with 10 ml/kg corn oil and fed the CA-1 diet, basophilic change of the proximal tubules was frequently observed, which is thought to be regeneration from injury or necrosis (Smith et al., 1972). Furthermore, chronic changes, i.e., fibrosis or the degeneration of the basement membrane in the proximal tubules, were not found in these kidneys. The three dams that showed severe changes in general condition were necropsied on day 1 of lactation, and the other dams were necropsied on day 3 or 4 of lactation. According to the above evidence, it is suggested that the necrosis of the proximal tubules occurred during the gestation period.
As severe damage of the kidney was not observed in nonpregnant female and male rats dosed with 10 ml/kg corn oil during 6 weeks and fed the CA-1 diet (unpublished data), pregnancy may be one of the factors increasing the toxicity of corn oil. During the late gestation period, rat fetuses are rapidly growing, and their nutritional requirements are increasing (Morriss et al., 1994). In this study, dams delivering relatively larger numbers of pups had more severe lesions of the kidneys among the dams treated with corn oil and fed the CA-1 diet. The nutrition required by a large number of fetuses may also exacerbate the corn oil toxicity in dams fed the CA-1 diet. The conditions observed in these dams resemble the ketotic state during pregnancy, which is characterized by fatty degeneration of the kidney (Smith et al., 1972
). In addition to this gestational burden, the stress of parturition and lactation may become a trigger for corn oil toxicity, thus causing its adverse effects to arise after parturition.
From the results of the autopsies of pups, it appears that corn oil may have no direct effects on development of fetuses and pups under the conditions of our study. The pup viability from dams treated with 10 ml/kg corn oil and fed the CA-1 diet was reduced; however, the exacerbation of the condition of these dams may have resulted in the reduction in the viability of pups.
In the safety evaluation of chemicals, adjustment of dosage volume by recently measured body weights is recommended. Because a fixed volume was applied during the gestation period in this study, the dosage volume was decreasing below the recommended volume as body weights of the pregnant rats were increasing. When dosage volume changes by recently measured body weight, administration of less than 10 ml/kg of corn oil in rats fed the CA-1 diet may raise the corn oil toxicity.
The mechanism of nephropathy occurring in the gestational and lactational rats in the present study remains to be elucidated. We would expect further experiments to resolve the remaining questions of nephrotoxicity by corn oil gavage in gestational and lactational rats, e.g., histopathologic examination of the kidneys of pregnant rats, the regulation of number of fetuses by hemisalpingectomy, or modification of ingredients in the diet, such as fiber. Thus, corn oil used as a vehicle may become a confounding factor in reproductive toxicity studies, depending on the diets fed to the experimental animals. Moreover, daily administration of 10 ml/kg of corn oil by gavage, especially to pregnant rats, is not recommended, even if corn oil is a convenient vehicle.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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