Comparative metabolism and kinetics of coumarin in mice and rats.

Coumarin, a well recognized rat hepatotoxicant, also causes acute, selective necrosis of terminal bronchiolar Clara cells in the mouse lung. Further, chronic oral gavage administration of coumarin at 200 mg/kg, a dose that causes Clara cell death, resulted in a statistically significant increased incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. In contrast, mouse lung tumors were not observed at the 100 and 50 mg/kg dose levels in the oral gavage study, or in CD-1 mice following chronic intake of coumarin at levels equivalent to 276 mg/kg in diet. The current studies were designed to determine the impact of oral gavage vs dietary administration on the pharmacokinetics and metabolism of coumarin in CD-1 and B6C3F1 mice and F344 rats. Following the administration of 200 mg/kg 14C-coumarin via oral gavage, lung C(max) values (total 14C-associated radioactivity) were five- and 37-fold greater than those resulting from a 50 mg/kg oral gavage dose or 1000 ppm in diet, respectively. Coumarin (200 mg/kg) pharmacokinetics and metabolism was also examined in F344 rats following oral gavage dosing. Total 14C-coumarin associated radioactivity in plasma was 3.5-fold lower than in the mouse, and the plasma half-life in rats was five-times longer than in mice. Using non-radiolabeled compound (200 mg/kg), coumarin and products of the coumarin 3,4-epoxidation pathway were quantitated in plasma and urine after oral gavage administration to mice and rats. 7-Hydroxycoumarin (7-HC) was quantitated in mouse plasma and urine. o-Hydroxyphenylacetic acid (o-HPAA) reached a concentration of 37 microg/ml in plasma, and accounted for 41% of the dose in the urine, whereas the C(max) for 7-hydroxycoumarin was 3 microg/ml, and represented 7% of the administered dose. In the rat, the plasma C(max) for o-HPAA was 6 microg/ml, and accounted for 12% of the dose. The coumarin C(max) in rat plasma was comparable to that in mouse. Coumarin 3,4-epoxide (CE) and its rearrangement product o-hydroxyphenylacetaldehyde (o-HPA) and o-hydroxyphenylethanol (o-HPE), were not detected at any time point in plasma or urine. This analysis of coumarin and CE pharmacokinetics in rodents suggests that the differential tumor response in the mouse oral gavage and dietary bioassays is a function of the route of exposure, whereas species differences in lung toxicity between mice and rats result from heightened local bioactivation in the mouse lung.

Urinary and bladder responses to immobilization in male rats.

Immobilization of groups of five to nine male rats for 2-5 days results in a 50% increase in urinary bladder fresh weight compared with normally caged controls. The increase in urinary bladder weight was not due to tissue oedema and was accompanied by epithelial hyperplasia in some urinary bladders. Immobilization did not alter total urine volume, but it did decrease the frequency of urine voiding and doubled the mean urine weight/voiding. Thus, bladder distention caused by the increased volume per voiding caused a rapidly induced increase in bladder tissue growth, and was accompanied by an increase in bladder epithelial cell division.

Comparison of the bladder response to indole and sodium saccharin ingestion by male rats.

To ascertain whether the bladder mass increase and epithelial hyperplasia induced by 5% dietary sodium saccharin (NaS) in short-term experiments with rats are caused by increased urinary excretion of indican associated with this treatment, the responses of the urine and bladder induced by 1.5% indole (Id) ingestion were compared with those induced by 5% NaS and 1.5% Id + 5% NaS. Id and NaS, when fed alone, produced equivalent increases in bladder mass and both compounds induced epithelial hyperplasia, but Id ingestion was associated with much greater urinary indican excretion (5 mg/g diet ingested) than was NaS (0.3 mg/g diet ingested). When Id and NaS were ingested together, the bladder mass increase was additive, but the epithelial hyperplasia was not exacerbated over that observed with each alone, and the urinary indican was equivalent to that produced by Id alone. These findings suggest that a high level of urinary indican excretion is associated with an increase in bladder mass and epithelial hyperplasia (Id treatment) but indicate that the relatively low urinary indican level obtained by NaS feeding alone is unlikely to be responsible for the bladder responses noted with this compound.

Comparison of the responses of male rats to dietary sodium saccharin exposure initiated during nursing with responses to exposure initiated at weaning.

In an attempt to define the role of exposure to sodium saccharin (NaS) during early life on the subsequent development of bladder tumours, we compared the responses of male rat pups to exposure to 5% dietary NaS initiated at parturition with those to exposure initiated at weaning. We also compared the effects of exposure from parturition to NaS given in a low-carbohydrate (L-CHO) diet with those of NaS in rat chow. NaS ingestion by the dam was associated with low saccharin concentrations in the pups' urine and had no effect on the caecal or bladder mass in the suckling pups. In the 10 wk after weaning, the rats ingesting NaS in chow showed decreased weight gain and increases in feed consumption, mass of caecal contents and tissue, urine output, bladder mass, relative water consumption (g water consumed/g feed consumed) and bladder hyperplasia. Except for bladder hyperplasia these effects were generally greater in the rats exposed to NaS from parturition than in those exposed only from weaning. The animals exposed to NaS in the L-CHO diet had the highest level of urinary saccharin but showed no bladder hyperplasia. The significance of these findings to the role of pre-weaning saccharin exposure in bladder tumorigenesis is discussed, and it is concluded that the effects on urinary parameters and the bladders of rats exposed to NaS during suckling and weaning may be secondary to the effects of NaS on the gastro-intestinal tract.

The effect of various saccharin forms on gastro-intestinal tract, urine and bladder of male rats.

Sodium saccharin, potassium saccharin, calcium saccharin and the free acid when fed to young male rats at a level of about 200 mumol/g diet all produced an equivalent increase in the caecal enlargement indicating that this phenomenon was due to the saccharin ion and not the accompanying cation. The sodium and potassium salts caused greater polydipsia and polyuria than the calcium or free acid forms. Simple hyperplasia of the bladder was noted in the rats ingesting the sodium and potassium salts but not in those ingesting the calcium or free acid forms. The difference in urine and bladder response to the salt forms is not attributable to the difference in the total urinary saccharin or the urinary concentration of saccharin. These results suggest that excess water absorption from the lower bowel and the concomitant bladder responses are dependent upon monovalent cation absorption but independent of saccharin absorption.

Effect of inherent urine output on the response of male rats to 7.5% dietary sodium saccharin.

Young male rats were preselected as high urine (57 g/kg body weight) or low urine (35 g/kg) voiders and were fed a diet containing 7.5% sodium saccharin (NaS) for 10 wk. Urine output was found to be a stable characteristic and high urine output was associated with increased water and feed consumption and increased weight gain. Rats responded in a very similar fashion to 7.5% dietary NaS regardless of their inherent urine output. NaS ingestion was associated with increases in water consumption, caecal mass and urine volume. Among rats that had ingested 7.5% dietary NaS for 10 wk there was a high incidence (12/20) of bladder epithelial hyperplasia. The results are discussed with regard to the concept that increased urine output is an important factor in NaS-induced bladder tumours.

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.

Effect of dietary carbohydrate type and content on the response of male rats to dietary sodium saccharin.

The role of dietary carbohydrate composition and concentration in the response of male rats to sodium saccharin (NaS) was ascertained by comparing the response to 5% dietary NaS in rats given diets containing 65% starch, 50% sucrose together with 15% starch, 65% glucose, or 3% sucrose. NaS induced similar levels of caecal enlargement and increases in urine volume and bladder mass when given with any of the three forms of carbohydrate at 65% in the diet. However with the 3% sucrose diet, NaS caused a lesser caecal enlargement and no increase in urine volume or bladder mass. These findings suggest that NaS not only inhibits saccharide hydrolysis but also inhibits glucose transport. The significance of these findings in relation to NaS-associated bladder tumours is discussed.

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.