Some Comments on the Selection of Human Intraspecies Uncertainty Factors

The Reference Dose (RfD) is used in the risk assessment of non-carcinogenic chemicals. It is derived by dividing a point of departure by the product of the uncertainty (UFs) and modifying factors (MFs). Separate UFs are used for different variables, e.g., intraspecies variation and, in general, each UF is an order of magnitude (10-fold). On the other hand, the MF is usually based on some known variable such as differences in absorption of a chemical from food and water and its default value is one. The USEPA's Integrated Risk Information System (IRIS) has 14 chemicals that have RfDs based on human studies. We examined those IRIS files to determine the rationale for setting human intraspecies uncertainty factors (UF_H). The first consideration was that the chemical had an adequate peer-reviewed human database. Without such, it would not be possible to derive an RfD based on human data. Ten of the 14 chemicals had an UF_H of 1 or 3; four of these were essential trace elements (ETEs). The rationales for using less than a 10-fold UF_H for the ETEs included; 1) nutritional data, 2) large human exposure groups, 3) minimal effect levels and/or 4) several studies with similar effect levels. For the other compounds, reasons included; 1) large human exposure groups, 2) a critical effect that was not adverse (cosmetic), 3) the most sensitive population was exposed, 4) the compound was on the FDA's “generally regarded as safe” (GRAS) list, 5) database uncertainties and 6) less-than-lifetime exposure adjusted for 70 years exposure. It is important to understand the reasons for selecting a UF_H of 1, or 3 as they will apply to future chemicals considered by the USEPA and other agencies.     

Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration

Bradyrhizobium japonicum cytochrome c ₅₅₀, encoded by cycA , has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c ₅₅₀ is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum , as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c ₅₅₀ is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c ₅₅₀ mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb ₃-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.     

Chlorate resistant mutants of Pseudomonas stutzeri affected in respiratory and assimilatory nitrate utilization and expression of cytochrome cd1

Abstract Chlorate-resistant mutants were generated by random insertion of the transposon Tn5 into genomic DNA of Pseudomonas stutzeri ZoBell strain and selected for loss of nitrate respiration (Nar phenotype). The mutants were differentiated by restriction-fragment analysis, by assaying for nitrate assimilation and for molybdenum co-factor activity, and by the amount of respiratory nitrate reductase. Two mutants, lacking both nitrate respiration and nitrate assimilation, over-produced an inactive nitrate reductase but synthesized in the presence of nitrate only a reduced amount of respiratory nitrite reductase (cytochrome cd ₁). Expression of cytochrome cd ₁ in these mutants was specifically induced by nitrate, suggesting a sensor system for this substrate.     

A new convenient method of azide condensation

Summary A new convenient modification of azide condensation is evaluated. The modification includes the use of tetrabutylammonium nitrite and p-toluene sulphonic acid, which are easily dissolvable in organic solvents, for the acidification of the reaction mixture.     

The oxidative damage and inflammation caused by pesticides are reverted by lipoic acid in rat brain

We have previously demonstrated that the administration of low doses of dimethoate, glyphosate and zineb to rats (i.p. 1/250 LD50, three times a week for 5 weeks) provokes severe oxidative stress (OS) in specific brain regions: substantia nigra, cortex and hippocampus. These effects were also observed in plasma. Lipoic acid (LA) is considered an “ideal antioxidant” due to its ability to scavenge reactive species, reset antioxidant levels and cross the blood–brain barrier. To investigate its protective effect we administered LA (i.p. 25, 50 and 100 mg/kg) simultaneously with the pesticide mixture (PM) for 5 weeks. After suppression of PM administration, we evaluated the restorative effect of LA for a further 5 weeks. LA prevented OS and the production of nitrites + nitrates [NOx] caused by PM in a dose-dependent manner. The PM-induced decrease in reduced glutathione and α-tocopherol levels in all brain regions was completely restored by LA at both high doses. PM administration also caused an increase in prostaglandins E₂ and F_2α in brain that was reduced by LA in a dose-dependent fashion. Taking into account the relationship between OS, inflammation and apoptosis, we measured caspase and calpain activity. Only milli- and micro-calpain isoforms were increased in the PM-treated group and LA reduced the activities to basal levels. We also demonstrated that interrupting PM administration is not enough to restore the levels of all the parameters measured and that LA is necessary to achieve basal status. In our experimental model LA displayed a protective role against pesticide-induced damage, suggesting that LA administration is a promising therapeutic strategy to cope with disorders suspected to be caused by OS generators, especially in brain.     

Mechanism of formate–nitrite transporters by dielectric shift of substrate acidity

Bacterial formate–nitrite transporters (FNTs) regulate the metabolic flow of small, weak mono-acids. Recently, the eukaryotic PfFNT was identified as the malaria parasite's lactate transporter and novel drug target. Despite crystal data, central mechanisms of FNT gating and transport remained unclear. Here, we show elucidation of the FNT transport mechanism by single-step substrate protonation involving an invariant lysine in the periplasmic vestibule. Opposing earlier gating hypotheses and electrophysiology reports, quantification of total uptake by radiolabeled substrate indicates a permanently open conformation of the bacterial formate transporter, FocA, irrespective of the pH. Site-directed mutagenesis, heavy water effects, mathematical modeling, and simulations of solvation imply a general, proton motive force-driven FNT transport mechanism: Electrostatic attraction of the acid anion into a hydrophobic vestibule decreases substrate acidity and facilitates protonation by the bulk solvent. We define substrate neutralization by proton transfer for transport via a hydrophobic transport path as a general theme of the Amt/Mep/Rh ammonium and formate–nitrite transporters.     

Effect of chilling on growth and nitrogen assimilation in Azolla caroliniana

Azolla caroliniana was exposed to 5 °C in darkness for 1, 2, 3, 5 or 7 d and then recovered for 7 d. Plants previously chilled for 2 or 3 d exhibited higher growth rates when transferred to normal temperature than either the control plants or those previously chilled for 5 or 7 d. Increased plant growth may be related to increased contents of chlorophyll, sucrose, and reducing sugars, due to increased photosynthetic capacity. In another experiment Azolla plants were chilled at 5 °C for 7 d and then transferred for 0, 4, 8, 12, or 16 d recovery to the N-free Hoagland solution or Hoagland solution containing 5 mM KNO₃. In previously chilled plants, the growth rate was decreased. In the medium supplemented with nitrogen, the growth rate was greater than in the N-free medium in both chilled and nonchilled plants. In chilled plants the decrease in growth rate may be related to the disturbance of Anabaena azollae cells where the protecting envelope of the heterocysts was deorganized. During the recovery the rate of N₂-fixation increased in both chilled and nonchilled plants up to 12 d after which both rates were similar. However, during the first 4 d the rate of the nonchilled plants was approximately 4-fold that of the previously chilled plants. Nitrate reductase and nitrite reductase activities in control plants were higher than in those previously chilled for 7 d. Both activities increased in nonchilled and previously chilled plants up to 12 d then decreased. The total protein content increased up to 12 d in chilled and nonchilled plants after which it decreased. Under all treatments, the values were higher in nonchilled plants than in those previously chilled ones and were also higher in presence of N than in its absence. Thus the presence of N-source in the medium counteracts the effect of chilling injury particularly during prolonged recovery.