F.R. Lefever

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.

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.

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.

Corticortophin releasing factor 2 receptor agonist treatment significantly slows disease progression in mdx mice

BackgroundDuchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy.MethodsUsing the mdx mouse model, we have evaluated the effect that corticotrophin releasing factor 2 receptor (CRF2R) agonist treatment has on diaphragm function, morphology and gene expression.ResultsWe have observed that treatment with the potent CRF2R-selective agonist PG-873637 prevents the progressive loss of diaphragm specific force observed during aging of mdx mice. In addition, the combination of PG-873637 with glucocorticoids not only prevents the loss of diaphragm specific force over time, but also results in recovery of specific force. Pathological analysis of CRF2R agonist-treated diaphragm muscle demonstrates that treatment reduces fibrosis, immune cell infiltration, and muscle architectural disruption. Gene expression analysis of CRF2R-treated diaphragm muscle showed multiple gene expression changes including globally decreased immune cell-related gene expression, decreased extracellular matrix gene expression, increased metabolism-related gene expression, and, surprisingly, modulation of circadian rhythm gene expression.ConclusionTogether, these data demonstrate that CRF2R activation can prevent the progressive degeneration of diaphragm muscle associated with dystrophin gene mutation.

Modifications of the human urocortin 2 peptide that improve pharmacological properties.

Recently, we demonstrated that the corticotropin releasing factor 2 receptor agonist, urocortin 2, demonstrated anti-atrophy effects in rodent skeletal muscle atrophy models. Compared to other CRF2R agonists however, the in vivo pharmacological potency of urocortin 2 is poor when it is administered by continuous subcutaneous infusion. Therefore, we attempted to modify the structure of urocortin 2 to improve in vivo efficacy when administered by subcutaneous infusion. By substituting amino acid residues in the linker region of urocortin 2 (residues 22-32), we have demonstrated improved in vivo potency without altering selectivity, probably through reduced CRFBP binding. In addition, attempts to shorten urocortin 2 generally resulted in inactive peptides, demonstrating that the 38 amino acid urocortin 2 peptide is the minimal pharmacophore.

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.

Activation of the dopamine 1 and dopamine 5 receptors increase skeletal muscle mass and force production under non-atrophying and atrophying conditions

BackgroundControl of skeletal muscle mass and force production is a complex physiological process involving numerous regulatory systems. Agents that increase skeletal muscle cAMP levels have been shown to modulate skeletal muscle mass and force production. The dopamine 1 receptor and its closely related homolog, the dopamine 5 receptor, are G-protein coupled receptors that are expressed in skeletal muscle and increase cAMP levels when activated. Thus we hypothesize that activation of the dopamine 1 and/or 5 receptor will increase skeletal muscle cAMP levels thereby modulating skeletal muscle mass and force production.MethodsWe treated isolated mouse tibialis anterior (TA) and medial gastrocnemius (MG) muscles in tissue bath with the selective dopamine 1 receptor and dopamine 5 receptor agonist SKF 81297 to determine if activation of skeletal muscle dopamine 1 and dopamine 5 receptors will increase cAMP. We dosed wild-type mice, dopamine 1 receptor knockout mice and dopamine 5 receptor knockout mice undergoing casting-induced disuse atrophy with SKF 81297 to determine if activation of the dopamine 1 and dopamine 5 receptors results in hypertrophy of non-atrophying skeletal muscle and preservation of atrophying skeletal muscle mass and force production.ResultsIn tissue bath, isolated mouse TA and MG muscles responded to SKF 81297 treatment with increased cAMP levels. Treating wild-type mice with SKF 81297 reduced casting-induced TA and MG muscle mass loss in addition to increasing the mass of non-atrophying TA and MG muscles. In dopamine 1 receptor knockout mice, extensor digitorum longus (EDL) and soleus muscle mass and force was not preserved during casting with SKF 81297 treatment, in contrast to significant preservation of casted wild-type mouse EDL and soleus mass and EDL force with SKF 81297 treatment. Dosing dopamine 5 receptor knockout mice with SKF 81297 did not significantly preserve EDL and soleus muscle mass and force although wild-type mouse EDL mass and force was significantly preserved SKF 81297 treatment.ConclusionsThese data demonstrate for the first time that treatment with a dopamine 1/5 receptor agonist results in (1) significant preservation of EDL, TA, MG and soleus muscle mass and EDL muscle force production during periods of atrophy and (2) hypertrophy of TA and MG muscle. These effects appear to be mainly mediated by both the dopamine 1 and dopamine 5 receptors.

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.

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.

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.