Extrapolation from in vitro tests to human risk: experience with sodium fluoride clastogenicity

Genotoxic effects observed in vitro, only at high doses or high levels of cytotoxicity, will be false positives if such conditions are not achieved or cannot be tolerated in vivo. However, for such effects to be disregarded there must be a threshold dose or level of cytotoxicity below which genotoxicity is absent. Sodium fluoride (NaF) has previously been shown to be clastogenic in vitro in Syrian hamster cells and human fibroblasts. We have extended these studies in human fibroblasts and included a positive control (mitomycin C, MMC) which is clastogenic in vivo and carcinogenic, and a chemically related control (NaCl). Cytotoxicity was measured as mitotic inhibition and cell death (loss of clonogenicity). The results are used to illustrate the problems associated with quantitative extrapolation from in vitro tests to human risk, as follows. (1) There appears to be a threshold response (clastogenicity vs. dose) with NaF at around 10 micrograms/ml (48 h exposure) but a more definitive conclusion must await elucidation of the mechanisms of clastogenicity. (2) NaCl is weakly clastogenic at 1000 times the threshold dose for NaF. The mechanisms are unlikely to be similar. (3) No clastogenicity was detected with NaF below about 30% mitotic inhibition but the relationship between clastogenicity and mitotic inhibition was similar for NaF and MMC. (4) There was no obvious threshold in the relationship between clastogenicity and cell killing with NaF. MMC was less clastogenic than NaF at equitotoxic doses. Observations 3 and 4 preclude the possibility of regarding the clastogenicity of NaF as a false positive by virtue of associated cytotoxicity.     

The nature and affinities of the “robust” Australopithecines: a review

The fossil evidence of the “robust” australopithecines is reviewed with an emphasis on the taxonomic divisions and evolutionary relationships among this group of hominids. The hypodigms of A. robustus, A. crassidens and A. boisei are described, and the significance of morphological variation within and between these species is assessed. Phylogenetic relationships among the “robust” australopithecines are examined using maximum parsimony analysis, and evolutionary scenarios are evaluated in the light of recent discoveries in East Africa.     

Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.     

Evolution of the Ribosomal DNA Spacers of Drosophila melanogaster : Different Patterns of Variation on X and Y Chromosomes

Length variation of the ribosomal gene spacers of Drosophila melanogaster was studied. Analysis of 47 X chromosomal and 47 Y chromosomal linked rDNA arrays collected from five continents indicates that the arrays on the two chromosomes differ qualitatively. The Y-linked arrays from around the world share little or no similarity for either their overall length or the organization of their spacers. Most of the X-linked arrays do, however, share a major length spacer of 5.1 kb. In addition, those X-linked arrays that have a major 5.1-kb band have similar spacer organization as demonstrated by genomic DNA digestions with several restriction enzymes. These data strongly support the hypothesis that spacer length patterns on only X-linked genes are maintained primarily by natural selection.     

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.     

Animal models in Q fever: pathological responses of inbred mice to phase I Coxiella burnetii

The susceptibility of inbred strains of mice to infection by phase I Coxiella burnetii, the aetiological agent of Q fever, was investigated by evaluating morbidity, mortality, antibody production and in vitro proliferative responses of splenic lymphocytes. Among the 47 strains of mice tested for morbidity and mortality to C. burnetii infection, 33 were resistant, 10 were of intermediate sensitivity, and four were sensitive. A/J mice exhibited the highest mortality, and surviving mice of this strain yielded high concentrations of viable rickettsiae from essentially all organs for more than 3 weeks after inoculation. However, A/J mice developed a protective immune response after vaccination with inactivated C. burnetii cells. Induction of gross pathological responses and antibody production were similar in sensitive mice (strain A/J) and resistant mice (strain C57BL/6J). The LD50 of phase I C. burnetii for A/J mice was about 1000-fold lower than that for the more resistant C57BL/6J mice. Mice of both strains developed antibody titres against phase I cells, phase II cells, and phase I lipopolysaccharide after the injection of one or more viable phase I organisms of C. burnetii; five or more rickettsiae caused splenomegaly that was almost proportional to the infecting dose. Suppression of in vitro proliferative responses of splenic lymphocytes to concanavalin A, a T-cell mitogen, was apparent after infection of sensitive A/J mice with as few as one to five phase I micro-organisms. However, suppression of proliferation of splenic lymphocytes from resistant C57BL/6J mice required 10(7) phase I C. burnetii.     

Lymphoma models for B cell activation and tolerance. VI. Reversal of anti-Ig-mediated negative signaling by T cell-derived lymphokines

We have recently described three "immature" B cell lymphomas which are exquisitely sensitive to growth inhibition by anti-Ig reagents and may serve as models for tolerance induction in normal B cells. These cells are inhibited from cell cycle progression into S after receiving a negative signal in early G1. In this paper, we demonstrate that the growth inhibition by anti-Ig can be prevented and reversed by the addition of supernatants from T cell lines. One such line, called Tova, produces factors which restore normal levels of DNA synthesis in the presence of concentrations of anti-Fab or anti-kappa immunoglobulins which cause up to a 90% inhibition of thymidine incorporation in a 2- to 3-day culture period. This factor is at least partially effective when added up to 24 hr after anti-Ig to unsynchronized lymphoma cells and it does not alter the growth of control cultures. Studies using synchronized lymphoma cells indicated that the T cell factor permitted cycle progression into S when added during the early G1 exposure to anti-kappa and was less effective when added late in G1. Preliminary characterization suggests that both B cell growth factor II (interleukin 5) and B cell stimulatory factor 1 (interleukin 4) have additive activity in this system, although another unidentified lymphokine may also be involved. The relevance of T cell reversal of Ig receptor-mediated negative signaling to neonatal B cell tolerance is emphasized.     

Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Km^r callus developing on hypocotyl sections. The totipotency of the Km^r callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Km^r tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Km^r callus and regenerated shoots.Abbreviations npt-II neomycin phosphotransferase II gene - NPT-II neomycin phosphotransferase II - nos nopaline synthase gene promoter - Km^r kanamycin resistant - BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MSD4×2 medium D4×2 based on Murashige & Skoog medium (see Scott & Draper, 1987)     

The isolation of a fetal rat liver glutathione S-transferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated from fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Yc (Mr 28,000) and a subunit (Mr 25,500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit 'Yfetus'. The enzyme which we term glutathione S-transferase YcYfetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.     

Gallbladder pressure, compliance, and hysteresis during cyclic volume change

The gallbladder has both storage and contractile properties, but is pressure-volume characteristics have not been fully described. We studied gallbladder pressure, compliance, stress relaxation, and the work performed during infusion-withdrawal of fixed volumes of bile at increments of base-line volume under halothane anesthesia in 13 dogs. Two cannulae were inserted in the gallbladder fundus: one for cyclic infusion-withdrawal of bile, and one for pressure monitoring. Following ligation of the cystic duct, the gallbladder was fully aspirated and then filled to successive predetermined base-line volumes (15, 20, 25, 30, or 35 mL). At each base-line volume, 5 mL of bile was infused and withdrawn at 2.47 mL/min. Studies were performed under basal conditions and with cholecystokinin octapeptide (CCK) infusion (10 or 30 ng.kg-1.h-1iv). Pressure was measured at base-line, after infusion, and after withdrawal. Compliance during infusion (delta V/delta P) was calculated for each cycle. Stress relaxation was defined as the difference between base-line pressure and any reduction in pressure after withdrawal. Hysteresis, the difference between work of infusion and work of withdrawal, was calculated. The results were such that base-line, end-infusion, and end-withdrawal pressures; work of infusion, work of withdrawal, and hysteresis; and stress relaxation all increased significantly with increases in the predetermined base-line volumes (p less than 0.001). Compliance decreased significantly (p less than 0.001) with increasing base-line volume. CCK at 10 ng.kg-1.h-1iv had no effect, but infusion of CCK at 30 ng.kg-1.h-1 significantly (p less than 0.05) increased pressure and work, decreased compliance, and increased stress relaxation compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)