Use of mechanistic information to derive chemical-specific adjustment factors - Refinement of risk assessment.

When extrapolating data from animal toxicological studies a default factor (dUF) of 100 is applied to derive a heath based guidance value. The UF takes into account the interspecies differences (ID) and the intraspecies variability (IV). When re-evaluating the safety of phosphates used as food additives nephrocalcinosis was identified as the critical endpoint. The underlying mechanism for nephrocalcinosis was attributed to the precipitation of calcium phosphate in the kidney, depending on its solubility, irrespective of the species and the population. Based on the mechanism, the volume of primary urine, for which the glomerular filtration rate (GFR) was used as a proxy, was considered to be the only parameter relevant for ID and IV. Median value of GFR in rats was 4.0 ml/min/kg bw. In humans it was 1.6 ml/min/kg bw in healthy adults and 0.9 in elderly. These values were calculated from the distribution of the GFR data from 8 studies in rats (n = 191), 16 studies in adults (n = 1540) and 5 studies in elderly (n = 2608). Multiplying the distribution of the ratio rat/healthy humans (ID) with the distribution of the ratio healthy humans/elderly human (IV) resulted in a phosphate specific factor of 4.5 (3.3-6.7) (median; 25th - 75th percentile).

Control by nutrients of growth and cell cycle progression in budding yeast, analyzed by double-tag flow cytometry.

To gain insight on the interrelationships of the cellular environment, the properties of growth, and cell cycle progression, we analyzed the dynamic reactions of individual Saccharomyces cerevisiae cells to changes and manipulations of their surroundings. We used a new flow cytometric approach which allows, in asynchronous growing S. cerevisiae populations, tagging of both the cell age and the cell protein content of cells belonging to the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The analysis of the findings obtained indicates that both during a nutritional shift-up (from ethanol to glucose) and following the addition of cyclic AMP (cAMP), two important delays are induced. The preexisting cells that at the moment of the nutritional shift-up were cycling before the Start phase delay their entrance into S phase, while cells that were cycling after Start are delayed in their exit from the cycle. The combined effects of the two delays allow the cellular population that preexisted the shift-up to quickly adjust to the new growth condition. The effects of a nutritional shift-down were also determined.

Fermentation of whey and starch by transformed Saccharomyces cerevisiae cells.

Among the main agro-industrial wastes, whey and starch are of prime importance. In previous work we showed that strains of Saccharomyces cerevisiae transformed with the episomal plasmid pM1 allow production of yeast biomass and ethanol from whey/lactose. Ethanol production from whey and derivatives has been improved in computer-controlled bioreactors, while fermentation studies showed that the composition of the medium greatly modulates the productivity (g ethanol produced/l in 1 h of fermentation). A yeast strain for the simultaneous utilization of lactose and starch has also been developed. Biotechnological perspectives are discussed.

A double flow cytometric tag allows tracking of the dynamics of cell cycle progression of newborn Saccharomyces cerevisiae cells during balanced exponential growth.

Studies on the dynamics of growth of single eukaryotic cells and their relationships with cell cycle regulations are generally carried out following cell synchronization procedures or, on a relatively low number of cells, by time-lapse studies. Establishment of both time-lapse studies and synchronous cell populations usually requires elaborate experimental efforts and is prone to perturb the physiological state of the cell. In this paper we use a new flow cytometric approach which allows, in asynchronous growing Saccharomyces cerevisiae populations, tagging of both the cell age and the cell protein content of a cohort of daughter cells at the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The experimental findings obtained indicate an exponential increase of the cell size during growth, that the daughter and the parent subpopulations grow with the same specific growth rate, that the average cell size increase rate of each individual cell is almost identical to the specific growth rate of the overall population and provide the opportunity to estimate the cell cycle length for the daughter cell population as well as the identification of the complex structure of asynchronously growing yeast populations.

Involvement of the alpha isoenzyme of protein kinase C in the growth inhibition induced by phorbol esters in MH1C1 hepatoma cells.

MH1C1 rat hepatoma cells express the alpha isoenzyme as the only phorbol-ester sensitive isoform of protein kinase C (PKC). In this cell line, phorbol 12-myristate 13-acetate (PMA) induced a marked, dose-dependent growth inhibition. The administration of the PKC inhibitor staurosporine was able to mimic the effect of the phorbol ester on cell growth in a dose-dependent fashion, whereas the PKC activator arachidonic acid stimulated cell proliferation. Exposure of cells to an antisense oligonucleotide specific for alpha PKC caused a significant impairment of cell growth. These data suggest that the alpha PKC activity is required for proliferation of MH1C1 cells.