An infant formula toxicity and toxicokinetic feeding study on carrageenan in preweaning piglets with special attention to the immune system and gastrointestinal tract.

A toxicity/toxicokinetic swine-adapted infant formula feeding study was conducted in Domestic Yorkshire Crossbred Swine from lactation day 3 for 28 consecutive days during the preweaning period at carrageenan concentrations of 0, 300, 1000 and 2250 ppm under GLP guidelines. This study extends the observations in newborn baboons (McGill et al., 1977) to piglets and evaluates additional parameters: organ weights, clinical chemistry, special gastrointestinal tract stains (toluidine blue, Periodic Acid-Schiff), plasma levels of carrageenan; and evaluation of potential immune system effects. Using validated methods, immunophenotyping of blood cell types (lymphocytes, monocytes, B cells, helper T cells, cytotoxic T cells, mature T cells), sandwich immunoassays for blood cytokine evaluations (IL-6, IL-8, IL1ß, TNF-α), and immunohistochemical staining of the gut for IL-8 and TNF-α were conducted. No treatment-related adverse effects at any carrageenan concentration were found on any parameter. Glucosuria in a few animals was not considered treatment-related. The high dose in this study, equivalent to ~430 mg/kg/day, provides an adequate margin of exposure for human infants, as affirmed by JECFA and supports the safe use of carrageenan for infants ages 0-12 weeks and older and infants with special medical needs.

Developmental toxicity studies in Crl:CD (SD) rats following inhalation exposure to trichloroethylene and perchloroethylene.

The potential for trichloroethylene (TCE) and perchloroethylene (PERC) to induce developmental toxicity was investigated in Crl:CD (SD) rats whole-body exposed to target concentrations of 0, 50, 150 or 600 ppm TCE or 0, 75, 250 or 600 ppm PERC for six hours/day, seven days/week on gestation day (GD) 6-20 and 6-19, respectively. Actual chamber concentrations were essentially identical to target with the exception of the low PERC exposure level, which was 65 ppm. The highest exposure levels exceeded the limit concentration (2 mg/L) specified in the applicable test guidelines. Maternal necropsies were performed the day following the last exposure. Dams exposed to 600 ppm TCE exhibited maternal toxicity, as evidenced by decreased body weight gain (22% less than control) during GD 6-9. There were no maternal effects at 50 or 150 ppm TCE and no indications of developmental toxicity (including heart defects or other terata) at any exposure level tested. Therefore, the TCE NOEC for maternal toxicity was 150 ppm, whereas the embryo/fetal NOEC was 600 ppm. Maternal responses to PERC were limited to slight, but statistically significant reductions in body weight gain and feed consumption during the first 3 days of exposure to 600 ppm, resulting in a maternal NOEC of 250 ppm. Developmental effects at 600 ppm consisted of reduced gravid uterus, placental and fetal body weights, and decreased ossification of thoracic vertebral centra. Developmental effects at 250 ppm were of minimal toxicological significance, being limited to minor decreases in fetal and placental weight. There were no developmental effects at 65 ppm.

Nitrite- and chloride-selective fluorescent nano-optodes and in vitro application to rat conceptuses.

Nitrite- and chloride-selective, ion correlation-based, nano-optodes have been prepared for application in vitro. These fluorescent, liquid polymer based sensors have theoretically predictable responses to anion activities and good selectivity. The nitrite sensor, prepared with a vitamin B12 derivative ionophore, would be useful for determination of oxidized nitric oxide. Chloride nano-optodes, as well as micro-optodes, prepared with an indium porphyrin ionophore, were utilized to determine chloride levels both on the surface and inside the visceral yolk sac of organogenesis-stage rat conceptuses.

Real time microfiberoptic redox fluorometry: modulation of the pyridine nucleotide status of the organogenesis-stage rat visceral yolk sac with cyanide and alloxan.

The surface of rat visceral yolk sacs (VYS) of intact, viable rat conceptuses were continuously monitored with a microfiberoptic sensor optimized for detection of the reduced pyridine nucleotides, NADH and NADPH. Model chemical toxins, cyanide and alloxan, were used and evaluated on the basis of their differential ability to modulate NAD(H)- and NADP(H)-dependent cellular pathways, respectively. Exposure with 2 mM sodium cyanide for 5 min caused a reversible fluorescence increase of 325 arbitrary fluorescence units (AFU) and 225 AFU on Gestational Days (GD) 10 and 11, respectively. Exposure with 40 mM alloxan for 5 min resulted in a fluorescence decrease of 170 and 120 AFU on GD 10 and 11, respectively. Glutathione (GSH) levels in the VYS, as determined by HPLC, showed a marked decrease from 27.3 +/- 2.1 to 2.9 +/- 0.4 pmol/mg protein, within the 5-min alloxan exposure period on GD 10. No decrease in GSH levels was noted for the same exposure duration on GD 11. A 2-hr pretreatment with 25 microM BCNU [(1,3 bis(2-chloroethyl)-1-nitrosourea], to inhibit glutathione disulfide reductase (GSSG-Rd), resulted in an elimination of the fluorescence decrease, but still led to a significant drop in GSH levels as seen on both days of gestation. These results are consistent with overall changes in intracellular pyridine nucleotide concentrations, where the relative amounts of NADPH increase significantly and disproportionately from GD 10 to 11. The net oxidation of NADPH, through GSSG-Rd activity, appears to be responsible for the alloxan-induced decrease in surface fluorescence. Conversely, the cyanide-induced fluorescence increases appear to be the result of NAD+ reduction, mediated through the inhibition of the terminal cytochrome oxidase in the electron transport chain.

Real time micro-fiberoptic monitoring of endogenous fluorescence in the rat conceptus during hypoxia.

A micro-fiberoptic methodology has been developed for non-invasive, real time measurement of endogenous pyridine nucleotide fluorescence from the surface of the visceral yolk sac (VYS) in intact, viable rat conceptuses. Gestational day (GD) 10-12 conceptuses are maintained in a customized perifusion system, which allows for control of oxygenation, as well as the continuous measurement of pH and oxygen concentration in the effluent perifusate. Miniaturized light guides were constructed by drawing 250 microns ESKA acrylic optical fibers through a stainless steel sheath with a high strength epoxy polymer. A single fiber supplied the excitation signal from a mercury arc lamp at a wavelength of 366 nm. The emission signal was returned via three additional fibers, electronically amplified, processed, and recorded, using a dual channel lamp-compensated fluorometer, optimized for detection of reduced pyridine nucleotides at 455 nm. Endogenous fluorescence in the conceptus was monitored by placing the polished tip of the sensor directly on the surface of the VYS. Oxygen-equilibrated conceptuses, exposed to 100% nitrogen, produced a reproducible biphasic surface fluorescence peak, which returned to baseline levels upon reoxygenation of the perifusate. This biphasic response consisted of an initial rapid rise in fluorescence (phase I), followed by an attenuated rate in fluorescence signal increase (phase II). The hypoxia produced age-dependent rates of fluorescence change during phase I, while phase II remained relatively unchanged throughout GD 10-12. These results demonstrate the ability to monitor endogenous fluorescence, non-invasively and in real time, during the period of organogenesis in the intact rat conceptus and will provide valuable information in studies of embryonic metabolism and response to chemical embryotoxicants.