Joseph J. Knapka

Contaminant and Nutrient Concentrations of Natural Ingredient Rat and Mouse Diet Used in Chemical Toxicology Studies

The NIH-07 open formula natural ingredient rat and mouse ration is the standard diet for chemical toxicity and carcinogenicity studies conducted for the National Toxicology Program (NTP). Contaminant and nutrient concentrations were determined in 2 to 94 lots of this diet used in the NTP toxicology studies. All nutrient concentrations were equivalent to or greater than the requirements for rats and mice as set forth by the National Research Council. Aflatoxins, Hg, chlorinated hydrocarbons except methoxychlor, organophosphates except malathion, estrogenic activity, and Salmonella sp. were not present at the detectable levels. Fluorine, As, Cd, Pb, Se, N-nitrosodimethylamine, N-nitrosopyrrolidine, N-nitrosomorpholine, nitrate, nitrite, butylated hydroxyanisole, butylated hydroxytoluene, ethylene dibromide, methoxychlor, malathion, and trypsin inhibitor activity were present at or above the detectable levels. Five lots of diet had nitrosamine content of 100 to 273 ppb and 7 lots had 2.08 to 3.37 ppm of Pb. All other lots of NIH-07 diet used for NTP toxicology studies contained low levels of the contaminants. After determination of the contaminant concentrations in the 94 lots of diet and the contaminant concentrations in natural ingredients used in formulating NIH-07 diet, maximum allowable levels of contaminants were established and a flexible scoring system for acceptability of each lot of diet for chemical toxicology studies was developed. By prescreening ingredients such as fish meal for heavy metals and nitrosamines, and applying the flexible scoring system proposed, more than 95% of the lots of NIH-07 diet produced during the last 3 years had scores of greater than or equal to 95 out of 100 points and were considered acceptable for toxicology studies.

Influence of oat bran of sucrose-induced blood pressure elevations in SHR

To determine whether oat fiber influences BP, we gave spontaneously hypertensive rats (SHR) a diet high in sucrose and low in protein (calories: sucrose 52%, protein 15%, fat 33%) or a diet low in sucrose and high in protein (calories: sucrose 13%, protein 52%, fat 35%). The amount of fat in these particular diets has not been shown to influence BP, so we modified the 2 diets by replacing fat with oat bran (10% w/w). Accordingly, we examined 4 groups of 5 rats consuming different diets: high sucrose, high sucrose + oat bran, low sucrose, and low sucrose + oat bran. Not unexpectedly, SHR consuming the diet high in sucrose had a significantly higher BP after 2 weeks than those consuming the diet low in sucrose. The significant difference in BP continued over the next 3 weeks. At the end of 6 week duration of study, we found the following BP: SHR ingesting the high sucrose diet, 217 mm Hg +/- 5 (SEM) vs SHR consuming the low sucrose diet, 187 mm Hg +/- 4 (SEM) p less than .0001]. SHR eating the low sucrose diet and consuming supplemental bran showed no significant change in BP after 6 weeks compared to SHR eating the basic diet alone, 188 mm Hg +/- 6 (SEM); however, 5 SHR consuming the high sucrose diet with added oat bran showed a significantly lower BP 200 mm Hg +/- 2 (SEM) than SHR ingesting the basic high sucrose diet devoid of oat bran [p less than .01]. We conclude that addition of oat bran to the diet can ameliorate sucrose-induced BP elevations in SHR.

Prevention of cataracts in pink-eyed RCS rats by dark rearing

Royal College of Surgeons rats have hereditary retinal degeneration and associated posterior subcapsular opacities (PSO) of the lens, detectable by slitlamp at 7-8 postnatal weeks in both pink- and black-eyed rats. The retinal degeneration is intensified by light, especially in pink-eyed rats. A fourth of pink-eyed rats developed mature cataracts by 9-12 months of age, but black-eyed rats whose retinas are protected from light by pigmented irises and pigment epithelium rarely have mature cataracts (3% or less), indicating light may be a factor in cataractogenesis. Prior work had shown that dark rearing reduced the rate of retinal degeneration in pink- but not black-eyed rats, but cataracts were not studied. In the present work, pregnant pink-eyed females were placed in a darkroom 1 week before parturition. Pups were removed over intervals at 20-85 postnatal days for: (a) microscopic study of fresh lenses and of fixed, stained retina and lens, and (b) counts of cells mm-2 of the web-like vitreous cortex after it had been dissected free. The macrophage-like cells are a quantitative index of immune reaction to retinal damage. At 50-53 postnatal days, in pink-eyed cyclic light reared RCS, the mean number of macrophages was 4.6-fold that in congenic controls, but in those that were dark reared it was only 1.4-fold. This was less than the increase in cyclic light reared black-eyed RCS (2.3-fold that in congenic black-eyed controls). Total absence of light reduced retinal degeneration and the number of macrophages, and prevented PSO detectable microscopically.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of excess sucrose ingestion on the life span of hypertensive rats (SHR)

Because dietary intake of sucrose may influence life span, we assigned 75 Spontaneously Hypertensive Rats (SHR) to be fed 5 diets, i.e., 15 rats were fed each diet. The control diet (I) derived near equal calories from sucrose, proteins, and fats. Diets II and III derived the majority of calories from sucrose with a decrease in calories from proteins (II) and fats (III) respectively. The last 2 diets were relatively low in sucrose with a higher percentage of calories from proteins (IV) and fats (V) respectively. Rats consuming the diets high in sucrose (II, III) had the shortest mean life span. Differences in renal function did not explain the shortened life span. Blood pressures in SHR ingesting high sucrose (II, III) were significantly higher, and longevity correlated with the mean BP of SHR. At post mortem, the majority of SHR showed enlarged hearts and pleural effusions. We conclude that the diets high in sucrose produced higher blood pressures with earlier congestive heart failure which is, at least in part, responsible for the shortened life span.

No effect of high sucrose diets on the kidneys of Wister Kyoto (WKY) rats

Dietary constituents, such as sucrose, may influence renal integrity during aging. Diets with high sucrose content cause extensive basement membrane damage in retinal vessels of WKY rats (J Clin Lab Sci 16: 419, 1986), which have been likened to the changes in diabetes mellitus and aging. Nevertheless, it is unknown whether similar lesions also occur in renal glomeruli and play a role in the progressive decline of renal function during aging. Knowledge of the latter possibility is important, because eye and renal vascular pathology often correlate, and earlier publications indicated that renal perturbations result from high sugar intake. In sharp contrast to work reported by others, our initial studies using Wistar rats showed no evidence of renal perturbations during months of high sucrose intake. We used diets formulated to be virtually the same in electrolyte and vitamin content, with the only differences being in the proportion of macronutrients — CHO (sucrose), fats and protein. To corroborate the initial studies, we expanded the studies using 40 unilaterally nephrectomized WKY rats. Despite marked differences in the consumption of macronutrients over a 6–7 month period, differences in kidney function and morphology were not seen among any of the 4 dietary groups. Although protein excretion was significantly higher in WKY rats consuming a low sucrose-high fat diet and BP was significantly higher in the rats consuming diets high in sucrose content, serum urea nitrogen and creatinine and urinary findings did not suggest renal damage secondary to high sucrose consumption. Light and electron microscopy failed to show renal morphological differences among the groups, similar to those reported in the vasculature of the eye. Based upon the ability of kidney slices to transport organic ions and consume OZ , renal tubular function also was not affected by different diets. We conclude that excess ingestion of sucrose over months causes no obvious renal perturbations in WKY rats, even though it elevates BP and causes eye lesions.

Environmental Factors in Cataractogenesis in RCS Rats

Royal College of Surgeons (RCS) rats have an inherited disease of the retinal pigmented epithelium (RPE), which shows a greatly reduced capacity to phagocytize the shed terminal portion of the outer segment of the rod photoreceptor cell (Mullen & LaVail, 1976). This failure in the symbiotic relationship of these two cells results in accumulation of outer segment membrane debris at the interface between the cells, and the lack of normal nutriture and membrane renewal leads to the death of all the rod photoreceptors by about 65 days in pink-eyed dystrophies and by 100 days in black-eyed dystrophies (LaVail & Battelle, 1975).