Carl L. Alden

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.

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.

Acute and subchronic nephrotoxicity of d-limonene in fischer 344 rats

In the studies described here, we have examined the sex-specific sensitivity of rat kidney to d-limonene. At 24 hr after an acute dose of 200 mg d-limonene/kg body weight administered to adult male and female Fischer 344 rats by oral gavage, an increase in the incidence and severity of hyaline droplets was observed in the kidneys of males only. This histological change was accompanied by a treatment-related increase in alpha 2u-globulin in males only and a greater accumulation of radioactivity in renal cortex of the male rat compared with that in the females dosed with [14C]d-limonene. In a separate subchronic study, groups of 5-wk-old male rats were administered d-limonene in a corn oil vehicle at 0 (control), 2, 5, 10, 30, or 75 mg/kg body weight by single daily gavage (5 days/wk) for 13 wk. Rats from selected dose groups received interim necropsies from days 8-29, while all groups were necropsied at the end of the study. Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg d-limonene/kg body weight. Histological examination of kidney tissue confirmed that d-limonene induced changes characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes collectively classified as chronic nephrosis. The no-observable-effect level for these effects was 5 mg d-limonene/kg body weight. At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at the 10 mg/kg body weight dose. It is concluded that treatment with d-limonene caused an increase in the formation of hyaline droplets in male rats only, that this increase was associated with an accumulation of alpha 2u-globulin, that d-limonene (or its metabolite) accumulated significantly in male rat kidney compared with that in females and that subchronic dosing produced a triad of morphological changes in the male rat kidney. These observations suggest that d-limonene caused nephrotoxicity specific to the male rat and that this toxicity may not be predictive of a similar response in humans.

Spontaneous Corneal Dystrophy and Generalized Basement Membrane Changes in Fischer-344 Rats

Groups of young, sexually mature Fischer-344 rats (n = 25/sex) obtained from 3 commercial breeders were examined ophthalmologically and histopathologically to determine the prevalence and severity of corneal basement membrane lesions (corneal dystrophy) and basement membrane changes in select nonocular tissues. Results disclosed a high incidence of corneal basement membrane dystrophy in rats of both sexes from all breeders; however, severity levels were significantly increased in rats obtained from one breeder when compared to others. Furthermore, rats that displayed the most advanced corneal lesions also exhibited more severe basement membrane changes in other organs, especially renal tubules and vascular internal laminae. These findings suggest that both ocular and nonocular dystrophic changes may have been linked through common physiologic (or genetic) mechanisms. Animals that displayed basement membrane lesions were not considered to represent compromised biologic test systems.

Increased hyaline droplet formation in male rats exposed to decalin is dependent on the presence of α2u-globulin

A peculiar decalin-induced male rat nephropathy associated with the altered renal handling of filtered protein appears limited to the accumulation of the protein, alpha 2u-globulin. Several strains of male rats that produce alpha 2u-globulin (Fischer-344, Sprague-Dawley, Buffalo, and Norway Brown) demonstrate spontaneous renal cortical hyaline droplets which are exacerbated after exposure to decalin. In all cases, a close correlation exists between hyaline droplet formation observed histologically and alpha 2u-globulin accumulation measured biochemically. In stark contrast, the NCI-Black-Reiter strain, which does not produce measurable quantities of alpha 2u-globulin, neither forms hyaline droplets nor accumulates any filtered protein in its kidney cortex either spontaneously or after exposure to decalin. Also, female rats injected ip with male rat alpha 2u-globulin exhibit increased hyaline droplet formation and alpha 2u-globulin accumulation when treated with decalin. These data provide evidence that the presence of alpha 2u-globulin is key in understanding why this nephropathy appears unique to the male rat.

Survey of the QSAR and in vitro approaches for developing non-animal methods to supersede the in vivo LD50 test

Quantitative structure-activity relationship (QSAR) studies and in vitro studies in which correlations with LD50 have been sought are reviewed. QSAR methods have shown some success in relating LD50 to certain physicochemical properties of the compound, particularly lipophilicity, but have been less successful in correlating LD50 with electronic properties of molecules (related to reactivity) or structural variables. It is concluded that insufficient evidence is available to determine whether QSAR methods can be of general use in predicting the acute toxicity (LD50) of chemicals, and that until further work is undertaken to develop QSARs for a much wider range of homologous series of compounds, this situation is unlikely to be resolved. New chemical descriptors that are more directly relevant to the mechanism of toxic action of the chemical should be identified. Cytotoxicity in vitro is poorly correlated with LD50, but good correlations have been obtained between toxicity in vivo and in vitro, using systems in which the toxic endpoint reflects the probable mechanism(s) of acute toxicity of the test chemical (e.g. the assessment of neurotoxins using neural cell systems). Therefore, it seems that the successful application of in vitro methods requires a better understanding of the mechanisms of acute toxicity in vivo and the development of mammalian cell culture systems that can model more closely the metabolic fate of the chemicals in vivo.

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.

A 20-month olestra feeding study in dogs

Three groups of beagle dogs (five/sex/group) were fed olestra, a mixture of octa-, hepta- and hexa-esters of sucrose formed with long-chain fatty acids, at 0, 5 or 10% of the diet for 20 months. The objective of the study was to assess the potential chronic toxicity of olestra in a non-rodent species. The feed was supplemented with vitamins A and E to ensure that the diets were nutritionally adequate and comparable for all groups. The levels of supplementation were established in a 91-day feeding study. Survival was 100% and growth was not affected by olestra. Olestra-fed animals consumed more feed than controls, apparently to compensate for the caloric dilution of the diet by olestra, but the increases were generally not statistically significant. No biologically significant changes were seen in haematological or serum biochemical parameters or in vitamin D and vitamin K status of the animals. Histopathology revealed no olestra-related effects. Isolated incidences of soft stools, apparently resulting from the large amounts of undigested olestra, were noted in olestra-fed animals. The results of this study indicate that olestra was not toxic when fed to dogs at up to 10% of the diet for 20 months.

Development of an Acute Model for the Study of Chloromethanediphosphonate Nephrotoxicity

Chloromethanediphosphonate (Cl2MDP), a cation chelator, is used as a therapeutic for hypercalcemia of malignancy. Cl2MDP exhibits nephrotoxic potential. Thus, a useful model has been developed to study the mechanism of injury. Intraperitoneal administration of highly exaggerated dosages, specifically 200 mg/kg b.i.d., resulted in a consistent mild to moderate extent of kidney damage after the third day of treatment in rats. Proteinuria and lowered serum phosphorus levels occur prior to onset of histopathologic changes. Injury was characterized as necrosis of proximal tubular epithelium with predilection for pars recta. Unlike many renal toxicity models, the necrosis occurs as cell lysis only after 24 to 48 hours of treatment. However, this model significantly reduces the time required to induce renal toxicity observed in routine toxicity studies from months of treatment to less than 1 week and will, thus, serve as a baseline for subsequent pathogenetic studies.

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.