A 90-day dietary study on kappa carrageenan with emphasis on the gastrointestinal tract.

Groups of Fischer 344 rats (20/sex/group) received control or treated diets at levels of 0, 25,000 or 50,000 ppm kappa carrageenan with a molecular weight range (Mw) of 196,000-257,000 Da for 90 days. The Low Molecular Weight Tail (LMT) ranged between 1.9% and 12.0%<50 kDa (mean 7%) based on the results of a program initiated to develop a validated analytical method to measure the LMT. This is the first GLP dietary study in which carrageenan is characterized by percentage LMT. Clinical examinations were performed daily. Individual food consumption/body weight measurements were made weekly. Ophthalmic exam was conducted prior to and at the end of treatment. Hematology/serum chemistry and urinalysis evaluations were done at necropsy, as were organ weight determinations for adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes and thyroid with parathyroids. Full histopathological evaluation of organs was conducted on the control and 50,000 ppm groups, including hematoxylin-eosin-stained cross sections of paraffin-embedded rolled colon. Clinical signs were limited to soft feces in high dose rats and to a lesser extent in low dose rats. There were no treatment-related effects on body weights, urinalysis, hematology or clinical chemistry parameters, or on organ weights or ophthalmic, macroscopic or microscopic findings. The gastrointestinal tract appeared normal in detailed histopathological evaluation using the Swiss roll technique. The NOAEL is 50,000 ppm in the diet (mean calculated test material consumption of 3394+/-706 mg/kg/day in males, 3867+/-647 mg/kg/day in females). The results of the study provide evidence that it is not necessary to characterize carrageenan by a specification for LMT (less than 5% below 50 kDa) as has been done in Commission Directive 2004/45/EC of 16 April 2004 (Commission Directive, 2004/45/EC of 16 April 2004 amending Directive 96/77/EC laying down specific purity criteria on food additives other than colors and sweeteners. Official Journal of European Union 20 April, 2004, L113/19-L113/21).

Cytoprotective properties of novel nonpeptide calpain inhibitors in renal cells.

Calpains are cytosolic, Ca(2+)-activated, neutral cysteine proteases. Rabbit renal proximal tubule (RPT) cells express both mu- and m-calpain. Although multiple calpain inhibitors protect against RPT cell death, most calpain inhibitors lack specificity, membrane permeability, and/or potency. A group of novel catalytic site-directed calpain inhibitors, including chloroacetic acid N'-[6,7-dichloro-4-(4-methoxy-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl]hydrazide (SJA7019) and chloroacetic acid N'-(6,7-dichloro-4-phenyl-3-oxo-3,4-dihydroquinoxalin-2-yl) hydrazide (SJA7029), were identified to be potent calpain inhibitors in vitro. The goals of this study were to determine the action of these two compounds on 1) RPT calpain activity using fluorescein isothiocyanate-casein zymography, 2) antimycin A-induced RPT extracellular (45)Ca(2+) influx and cell death, and 3) hypoxia/reoxygenation-induced RPT cellular dysfunction and death. The results showed that the SJA compounds inhibited RPT mu- and m-calpain with equal potency (approximate IC(50), 30 microM) and efficacy, and blocked antimycin A-induced extracellular Ca(2+) influx and cell death. In addition, SJA7029 blocked cell death and allowed the recovery of mitochondrial function and active Na(+) transport in RPTs subjected to hypoxia/reoxygenation. In summary, the SJA compounds 1) were more potent inhibitors of calpains than catalytic site-directed peptide inhibitors in this model, 2) prevented extracellular Ca(2+) influx during the late phase of cell death, and 3) are true cytoprotectants and allow recovery of RPT cellular functions after injury.