Toxicological evaluation of methyl tertiary butyl ether (MTBE): Testing performed under TSCA consent agreement

MTBE is a colorless, relatively volatile liquid that has found widespread use as an octane‐enhancing gasoline additive. In 1987, the Environmental Protection Agency's (EPA) Interagency Testing Committee identified MTBE for priority testing consideration based on large production volume, potential widespread exposure, and limited data on chronic health effects. In response, the industry formed the MTBE Health Effects Testing Task Force, which in 1988 signed a Consent Agreement with the EPA requiring the task force member companies to perform toxicological testing on MTBE.

The testing program, which began in the second quarter of 1988, consists of a full complement of short‐ and long‐term tests. The testing completed to date includes genotoxicity (in vivo bone marrow cytogenetics and Drosophila sex‐linked recessive lethal assays), developmental toxicity, acute and subchronic neurotoxicity (motor activity, functional observation battery, and neuropathology), subchronic toxicity, reproductive/fertility effects, and pharmacokinetic studies. There is also an ongoing oncogenicity study in rats and mice. The final report for this chronic study is expected at the end of 1992. The total cost for the program is approximately $3.75 million, which is funded by the 11 Task Force member companies based on market share.

These studies were sponsored by the MTBE Health Effects Testing Task Force, Oxygenated Fuels Association, Washington, D.C.     

Positive residues involved in the voltage-gating of the mitochondrial porin-channel are localized in the external moiety of the pore.

The role of positive charges located on the hydrophilic surface of the mitochondrial outer membrane channel was investigated by studying the interaction between LDAO-solubilized porin and a cation-exchanger column. The binding of porin to the column material was inhibited when the elution buffer had a pH of 9 or when 2 mM dextran sulfate was added to the buffer at neutral pH. Interestingly, the addition of a synthetic copolymer of methacrylate, maleate and styrene known as a potent modulator of the voltage-dependence, did not influence the interaction between column material and porin. Incubation of porin with fluorescein isothiocyanate (FITC) resulted in the isolation of a porin fraction in which on average two lysines located on the surface of the pore-forming complex per 35 kDa polypeptide were modified. The voltage-dependence of the fluorescein isothiocyanate modified porin was strongly decreased as compared with the unmodified porin. The experiments presented here give the first biochemical evidence that positively charged lysine residues located on the surface of the channel-forming complex are responsible for the gating of the mitochondrial porin-channel.     

Variation at the apolipoprotein (apo) AI-CIII-AIV gene cluster and apo B gene loci is associated with lipoprotein and apolipoprotein levels in Italian children.

We have used RFLPs of the apolipoprotein (apo) B gene and apo AI-CIII-AIV gene cluster to estimate the genetic contribution of variation at these loci to the variability of plasmid lipid, lipoprotein, and apolipoprotein levels in 209 children from Sezze in central Italy. The sample was randomly divided into group I (107 children) and group II (102 children). Four site polymorphisms (PvuII, XbaI, MspI, and EcoRI) of the apo B gene and five site polymorphisms (XmnI, PstI, SstI, PvuII-CIII, and PvuII-AIV) of the apo AI-CIII-AIV gene cluster were examined in group I children. After adjustment for gender, age, and body-mass index, polymorphisms at both gene loci (PvuII-B, PvuII-CIII, and PvuII-AIV) were associated with significant effects on the levels of plasma apo AI, apo B, or high-density lipoprotein-cholesterol. RFLPs that showed significant effects in group I were genotyped in group II. All three polymorphisms were associated with similar effects on apolipoprotein levels, though for all RFLPs the magnitude of the effects was smaller in the group II children and only statistically significant for the effect of the PvuII-B genotype on apo AI levels. In the total sample of 209 children 7.4% of the sample variance in apo AI levels was explained by variation associated with the apo B PvuII-B RFLP. In addition, the PvuII-B RFLP was associated with significant effects on plasma apo B levels and explained 5.7% of the sample variance. The PvuII-CIII and PvuII-AIV polymorphisms were both associated with differences in apo AI levels, explaining 3.7%-5.7% of the sample variance. Taken together, the three PvuII polymorphisms explained 17.7% of the phenotypic variance in apo AI levels. There was significant evidence for an effect of nonlinearity of the PvuII-CIII genotypes on apo AI levels, with the individuals heterozygous for the polymorphism having the highest apo AI levels. No evidence of interaction between genotype and gender, age, and body-mass index was shown by covariance analysis. The molecular explanation of this effect is unclear. Our data show that variation at both the apo AI-CIII-AIV and apo B loci are associated with lipoprotein and apolipoprotein levels in this sample of Italian children.     

Characterization of a Manganese Superoxide Dismutase from the Higher Plant Pisum sativum

A manganese-containing superoxide dismutase (EC 1.15.1.1) was fully characterized from leaves of the higher plant Pisum sativum L., var. Lincoln. The amino acid composition determined for the enzyme was compared with that of a wide spectrum of superoxide dismutases and found to have a highest degree of homology with the mitochondrial manganese superoxide dismutases from rat liver and yeast. The enzyme showed an apparent pH optimum of 8.6 and at 25°C had a maximum stability at alkaline pH values. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by pea leaf manganese superoxide dismutase was found to be 1.61 × 10⁹ molar⁻¹·second⁻¹ at pH 7.8 and 25°C. The enzyme was not sensitive to NaCN or to H₂O₂, but was inhibited by N₃⁻. The sulfhydryl reagent p-hydroxymercuribenzoate at 1 mm concentration produced a nearly complete inhibition of the manganese superoxide dismutase activity. The metal chelators o-phenanthroline, EDTA, and diethyldithiocarbamate all inhibited activity slightly in decreasing order of intensity. A comparative study between this higher plant manganese superoxide dismutase and other dismutases from different origins is presented.     

Cultivation of formerly noncultivable strains ofMycoplasma hyorhinis

Growth on axenic agar medium is one of several characters by which mycoplasmas are defined. In apparent contradiction of the definition, DBS 1050 and other noncultivable strains ofMycoplasma hyorhinis do not grow on axenic medium but grow in cell culture. Our results show that BHK-21 cell extracts support DBS 1050 growth in appropriate medium. An inhibition assay, based on a virus neutralization format, shows that a variety of common medium ingredients inhibit DBS 1050 growth. The most potent activity was found in yeast extract. All other noncultivable strains ofM. hyorhinis tested have a yeast extract sensitivity, while cultivable strains do not. The apparent cell dependence of DBS 1050 can be attributed to growth inhibition due to factors present in a wide variety of peptones and extracts commonly used in medium; preferential growth in cell cultures is due to the absence of effective levels of these factors. Data are not available to determine if cell cultures provide growth factors not found in standard medium. The infraspecific taxon,M. hyorhinis cultivar α, is proposed for formerly noncultivable strains ofM. hyorhinis.     

Bombesin production by human small cell carcinoma of the lung

A series of continuous cell lines of human small cell carcinoma of the lung (SCCL) have been evaluated for the production of bombesin (BN). In early established cultures BN was detected in the medium of 9 out of 11 cell lines and in 6 out of 7 cell homogenates examined. Levels in the medium were frequently higher in cultures of later passages compared to earlier passages of the same line and low levels developed in the two previously negative cell lines. Plasma concentrations were greater than 80 pmol/l in 2 out of 27 (7%) randomly selected patients with SCCL. A culture (DMS 406) established from the tumor of a patient with the highest plasma level (1240 pmol/l) was the highest producer in vitro. The results indicate that BN, which has been demonstrated immunocytochemically to be present in normal bronchial mucosal cells, is frequently produced by SCCL in vitro but elevated plasma levels are infrequently found in patients with this neoplasm.     

Restriction enzyme analysis of the β-globin gene in DNA from β°-thalassaemic subjects from Ferrara

The map of restriction sites including and surrounding the δ- and β-globin genes has been established for three Ferrara β°-thalassaemic subjects. The fragments obtained using nine restriction enzymes do not show any differences from normal DNA. Among others, restriction enzymes giving short fragments at the 5′ and 3′ ends of the β-globin structural gene have been employed. The results obtained for the thalassaemic DNA are identical to those for control DNA, thus excluding the presence of extensive deletions in or adjacent to the coding regions of the β-globin gene in Ferrara β°-thalassaemia.     

Selective exposure of mucopolysaccharides is involved in macrophage physiology.

Surface and intracellular mucopolysaccharides of guinea-pig peritoneal macrophages maintained in suspension and monolayer culture were studied. At least five classes of compound (hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate and chondroitin 6-sulfate) were resolved and characterized by electrophoresis and enzymatic degradation. The results reported here suggest that modulation of mucopolysaccharide exposure is involved in macrophage physiology. The possible biological role of surface mucopolysaccharides in macrophage activity is discussed.     

Flash-induced photophosphorylation in Rhodospirillum rubrum chromatophores I. The relationship between cytochrome c-420 content and photophosphorylation

The content of cytochrome c-420 in Rhodospirillum rubrum chromatophores prepared by grinding with alumina is 5–10% of that in whole cells, and 20–40% in chromatophores by ‘French’ pressing.Flash-induced phosphorylation of various chromatophores which varied in cytochrome content from 7 to 40% is proportional to the cytochrome content. Extrapolating the cytochrome c-420 content to that observed in whole cells, a ratio $\text{ATP}\text{P}{}^{\mathrm{+}}\text{X}{}^{\mathrm{?}}$ near 1 is calculated. At low flash intensity the phosphorylation per flash is proportional to flash energy.Photophosphorylation in flashes given after a time of several minutes is only slightly dependent on the number of flashes. If the flashes are spaced from 0.1 to 10 s, relative phosphorylation in the first flash is about 70% and in the second 90% of that observed in the following flashes. Proton binding is not affected by the cytochrome c-420 content and a ratio of $\text{H}{}^{\mathrm{+}}\text{P}{}^{\mathrm{+}}\text{X}{}^{\mathrm{?}}$ of 2.3 was found.These results can be explained by a working hypothesis in which charge separation occurring at one reaction centre and the resulting electron transport mediated amongst others by c-420, results in the injection of two protons into an ATPase, this in contrast to a chemiosmotic mechanism, where the protons are released in the chromatophore inner space.