Cenozoic evolution of deep ocean temperature from clumped isotope thermometry.

Characterizing past climate states is crucial for understanding the future consequences of ongoing greenhouse gas emissions. Here, we revisit the benchmark time series for deep ocean temperature across the past 65 million years using clumped isotope thermometry. Our temperature estimates from the deep Atlantic Ocean are overall much warmer compared with oxygen isotope-based reconstructions, highlighting the likely influence of changes in deep ocean pH and/or seawater oxygen isotope composition on classical oxygen isotope records of the Cenozoic. In addition, our data reveal previously unrecognized large swings in deep ocean temperature during early Eocene acute greenhouse warmth. Our results call for a reassessment of the Cenozoic history of ocean temperatures to achieve a more accurate understanding of the nature of climatic responses to tectonic events and variable greenhouse forcing.

InterCarb: A Community Effort to Improve Interlaboratory Standardization of the Carbonate Clumped Isotope Thermometer Using Carbonate Standards.

Increased use and improved methodology of carbonate clumped isotope thermometry has greatly enhanced our ability to interrogate a suite of Earth-system processes. However, interlaboratory discrepancies in quantifying carbonate clumped isotope (Δ47) measurements persist, and their specific sources remain unclear. To address interlaboratory differences, we first provide consensus values from the clumped isotope community for four carbonate standards relative to heated and equilibrated gases with 1,819 individual analyses from 10 laboratories. Then we analyzed the four carbonate standards along with three additional standards, spanning a broad range of δ47 and Δ47 values, for a total of 5,329 analyses on 25 individual mass spectrometers from 22 different laboratories. Treating three of the materials as known standards and the other four as unknowns, we find that the use of carbonate reference materials is a robust method for standardization that yields interlaboratory discrepancies entirely consistent with intralaboratory analytical uncertainties. Carbonate reference materials, along with measurement and data processing practices described herein, provide the carbonate clumped isotope community with a robust approach to achieve interlaboratory agreement as we continue to use and improve this powerful geochemical tool. We propose that carbonate clumped isotope data normalized to the carbonate reference materials described in this publication should be reported as Δ47 (I-CDES) values for Intercarb-Carbon Dioxide Equilibrium Scale.

Assaying the antioxidant and radical scavenging properties of aliphatic mono- and di-N-oxides in superoxide dismutase-deficient yeast and in a chemiluminescence test.

The antioxidative action of amphiphilic mono-(alkanoylamino) ethyldimethylamine-N-oxides (EDA), di-N-oxides 1,1-bis {[2-(N,N-dimethylamino)ethyl]amido}alkane-di-N-oxides (MEDA) and 1,1-bis {[3-(N,N-dimethylamino)propyl]amido}alkane-di-N-oxides (MPDA) with a 12- and 14-membered acyl chain against tert-butylhydroperoxide (TBHP)-produced peroxyl and paraquat (PQ)-generated superoxide radicals was determined in superoxide dismutase-deficient mutants of Saccharomyces cerevisiae, and, in parallel, in a chemical assay based on chemiluminescence changes caused in a luminol system by peroxyl radicals generated from the azo-compound 2,2'-azobis(2-amidinopropane dihydrochloride) (AAPH). At 30 micromol/L, the shorter-chain compounds did not affect strain survival while longer-chain ones, in some cases, lowered the survival of sod2 and sod1 sod2 cells. Whether nontoxic or medium-toxic, all N-oxides protected the sod strains against the toxic effect of PQ and TBHP, the protection being stronger with the di-N-oxides. The survival was lowered only by 14-MPDA in the TBHP-exposed sod2 mutant. Membrane lipids isolated from all strains were protected against TBHP-induced peroxidation by both mono- and di-N-oxides, the protection being dependent on the alkyl chain length. Mono-N-oxides were again less active than di-N-oxides with the same alkyl chains, the antiperoxidative activity being also dependent on lipids isolated from the individual mutants. In the chemiluminescence assay, the IC50 value of the N-oxides for scavenging of radicals generated from AAPH generally decreased (i.e. the scavenging efficiency increased) with increasing chain length and was the highest in MEDA.

Amphiphilic amine-N-oxides with aliphatic alkyl chain act as efficient superoxide dismutase mimics, antioxidants and lipid peroxidation blockers in yeast.

Amphiphilic 3-(alkanoylamino)propyldimethylamine-N-oxides with different length of the alkyl chain, i.e. different hydrophilic-lipophilic balance, act in micromolar concentrations as SOD mimics by lifting the inhibition of aerobic growth caused by SOD deletions in Saccharomyces cerevisiae. They also enhance the survival of sod mutants of S. cerevisiae exposed to the hydrophilic superoxide-generating prooxidant paraquat and the amphiphilic hydroperoxide-producing tert-butylhydroperoxide (TBHP), and largely prevent TBHP-induced peroxidation of isolated yeast plasma membrane lipids. Unlike the SOD-mimicking effect, the magnitude of these effects depends on the alkyl chain length of the amine-N-oxides, which incorporate into S. cerevisiae membranes, causing fluidity changes in both the hydrophilic surface part of the membrane and the membrane lipid matrix. Unlike wild-type strains, the membranes of sod mutants were found to contain polyunsaturated fatty acids; the sensitivity of the mutants to lipophilic pro-oxidants was found to increase with increasing content of these acids. sod mutants are useful in assessing pro- and antioxidant properties of different compounds.

Cell transformation and genotoxicity induced by bis(2, 3-dichloro-1-propyl) ether.

Bis(dichloropropyl) ether isomers have been identified in a petrochemical plant effluent through a toxicity identification evaluation study in the United States. They have also been observed in the microgram per liter range along one of the largest rivers in Europe, the Elbe River. In the present investigation, the genotoxic and transforming activity of a bis(dichloropropyl) ether isomer, bis(2,3-dichloro-1-propyl) ether, was assayed in vitro. The results demonstrate that bis(2,3-dichloro-1-propyl) ether is a potent mutagen in Salmonella typhimurium strains TA 100, TA 1535, and to a lesser extent in strain TA 98, but only when tested in the presence of a metabolic activation system (S9 mix). We have also investigated the induction of micronuclei by bis(2,3-dichloro-1-propyl) ether in the metabolically competent cell line, MCL-5. A linear, dose-dependent increase in micronuclei was observed following exposure to bis(2,3-dichloro-1-propyl) ether. The DNA strand-breaking capacity of this chemical was assessed in the alkaline single-cell gel electrophoresis ("comet") assay with MCL-5 cells. Bis(2,3-dichloro-1-propyl) ether clearly induced DNA strand breaks in the 4.5-45.5 microg/ml dose range. The ether also induced malignant transformation in C3H/M2 mouse fibroblasts after metabolic activation (S9 mix). Thus, it must be suspected that bis(2, 3-dichloro-1-propyl) ether may possess a carcinogenic potential. Since the compound along with its isomers is present in considerable concentrations in surface water, their elimination is a matter of significant public concern.

Synthesis, Surface Properties, and Hydrolysis of Chemodegradable Anionic Surfactants: Diastereomerically Pure cis- and trans-2,5-Disubstituted-1,3-dioxanes

In our continuing systematic studies concerning the synthesis, surface properties, and hydrolysis of chemodegradable, diastereomerically pure surface active 2,5-disubstituted 1,3-dioxane derivatives, two new groups of anionic surfactants, sodium cis- and trans-(2-n-undecyl-1,3-dioxan-5-yl)methyl sulfates and sodium cis- and trans-3-[(2-n-undecyl-1,3-dioxan-5-yl)oxy]propanesulfonates, were synthesized and investigated. Surface properties of these surfactants, i.e., surface excess concentration, Gamma, surface area demand per molecule, A, effectiveness of surface tension reduction, Pi, critical micelle concentration, CMC, and standard free energies of adsorption, DeltaGads0, and of micellization, DeltaGmic0, were determined. It was shown that the trans-isomers, in which the configuration of the polar group is equatorial, are more surface active than the cis-isomers with axial configuration of the polar group at the C-5 carbon atom of the 1,3-dioxane ring. The surfactants under study undergo easy hydrolysis reaction in DCl/D2O solution with cleavage of the 1,3-dioxane ring to nonsurface active intermediates. The trans-isomers are hydrolyzed faster than cis-isomers.

Malignant transformation of mouse M2-fibroblasts by glycerol chlorohydrines contained in protein hydrolysates and commercial food.

Glycerol chlorohydrines, such as 3-chloro-1,2-propanediol and 1,3-dichloro-2-propanol, are present in commercial protein hydrolysates used for human nutrition. These compounds are genotoxic and 1,3-dichloro-2-propanol induced tumors in rats. Now it is reported that both compounds are active at inducing malignant transformation of mouse fibroblasts. Therefore, the carcinogenic risk to humans by exposure to these compounds contained in food is of concern. The investigation of the in vivo carcinogenic potential of 3-chloro-1,2-propanediol is urgently required to further evaluate the carcinogenic risk to exposed consumers.

Proliferation of fetal rat hepatocytes in response to growth factors and hormones in primary culture.

Fetal rat hepatocytes (day 19 of gestation) multiply in primary culture in arginine-free, hydrocortisone-containing chemically defined medium MX-82 supplemented either with epidermal growth factor (EGF) or insulin or both. In contrast, hepatocytes did not multiply under similar culture conditions using Dulbecco's minimum essential medium (DMEM). Cells underwent two divisions within 10 days in cultures maintained in MX-82 medium without a medium change, and cells grew to increased final cell densities when the medium was renewed every third day. When the medium MX-82 was enriched by the addition of lipids, intermediary metabolites, and trace metals (medium MX-83), cells grew to higher densities. In the absence of the growth factors, cells became quiescent and subsequently could be induced to synthesize DNA in response to EGF. With the increasing numbers of cells per dish, the growth response of the hepatocytes diminished. Levels of hepatocyte-specific albumin and alpha-fetoprotein mRNAs at day 0 were similar to those observed at day 10 in primary fetal rat hepatocyte cultures and were maintained at higher levels in medium MX-83 than in medium MX-82.

Altered growth factor requirements and cell cycle control in rat hepatoma cells versus adult rat hepatocytes in culture.

Adult rat hepatocytes multiply in primary cultures when incubated in arginine-free MX-83 medium supplemented with dialyzed fetal calf serum, insulin, glucagon, hydrocortisone, epidermal growth factor, and transferrin. In the absence of mitogens, the fraction of the cells engaged in DNA synthesis dropped sharply. However, cells initiated DNA synthesis in response to the mitogenic mixture indicating that hepatocyte proliferation is controlled by G1----S transition rates. In contrast, rat hepatoma line DTH-3, derived from Morris 7777 "minimal deviation" hepatoma, required only insulin for proliferation in chemically defined MX-83 medium. The lengths of their cell cycle phases varied with the growth rate. The phases of the growth cycle were proportionately shortened (expanded) when the growth rate was increased (decreased). It is concluded that DTH-3 hepatoma cells, which display a decreased growth factor requirement as compared with adult rat hepatocytes differ from normal hepatocytes by fundamental alterations in the mechanisms controlling the progression of the cell cycle.

Immortalized differentiated hepatocyte lines derived from transgenic mice harboring SV40 T-antigen genes.

Hepatocytes of transgenic mouse fetuses harboring SV40 virus transforming gene sequences in the SV delta e-MGH fusion gene construct 202 driven by the mouse metallothionein (MT-I) enhancer [R. D. Palmiter, H. Y. Chen, A. Messing, and R. L. Brinster (1985) Nature (London) 316, 457-460] were cultured at Day 19 of gestation and established as a differentiated line expressing albumin and alpha-fetoprotein (AFP) mRNAs. Hepatocyte line FMH-202 contains integrated SV40 sequences, expresses SV40 T-antigen genes, and exhibits unlimited growth potential because it has been cultured 18 months without apparent decrease in cell viability or in growth rate that could suggest the occurrence of a crisis period. Immortalized cells multiply in chemically defined medium deficient in arginine with transferrin plus insulin, whereas EGF, insulin, and transferrin are obligatory requirements for fetal or newborn mouse hepatocyte multiplication in primary cultures. Cells did not grow in agar and were not tumorigenic in nude mice. Their immortalized, nonmalignant phenotype was further documented by low saturation densities of confluent monolayers showing no overgrowth, and by growth arrest in the absence of insulin with subsequent induction of DNA synthesis and resumption of cell growth in response to insulin. Thus, it appears that immortalized SV40 T-antigen-expressing hepatocytes are present in the liver of the transgenic mice. However, at later points in liver development the transforming activity of T-antigen becomes apparent and leads to hepatocellular carcinoma formation in vivo.

Transformation of differentiated neonatal rat hepatocytes in primary culture by polyoma virus early region sequences.

Neonatal rat hepatocytes (NRH) in primary cultures were transformed upon transfection with plasmid pSV 5-neo containing polyoma virus (Py) early region sequences. Clones of cells (Py-NRH) resistant to the antibiotic G418 were obtained after selection in arginine-deficient medium with serum, epidermal growth factor (EGF), transferrin and insulin. They did not undergo senescence during serial subcultivation. One clone (Py-NRH Cl A) harbored a single integrated copy of Py early region sequences and expressed transforming Py genes, hepatocyte-specific transcripts, including albumin, alpha-fetoprotein (AFP) and tyrosine aminotransferase (TAT) mRNAs. Subclones isolated after about 45 cell doublings still contained albumin and AFP, but no TAT mRNAs, indicating that long-term stabilization of liver functions is not necessarily permanent unless selected for (e.g. arginine synthesis). Cells grew unrestricted in medium containing insulin and no longer required EGF. Cells grew in agar, secreted a beta-transforming growth factor-like activity into the medium and were tumorigenic in nude mice. Hybridization studies using v-erbB DNA as a probe showed that Py-NRH, unlike neonatal hepatocytes in primary culture, express the EGF receptor gene at low or undetectable levels. Py-NRH Cl A and a subclone (5A) derived from it, however, contained elevated levels of rat c-neu oncogene-related RNA, whereas levels in another subclone (3A) were low or undetectable. These findings demonstrate that a proto-oncogene was activated after transfection of hepatocytes with DNA tumor virus transforming genes. However, the expression of c-neu oncogene is not related to the maintenance of the transformed state.

Rat platelets contain growth factor(s) distinct from PDGF which stimulate DNA synthesis in primary adult rat hepatocyte cultures.

Adult rat hepatocytes in primary cultures are stimulated to synthesize DNA in response to rat serum, whereas rat plasma is considerably less active. Biological activity is present in rat platelets and is secreted during aggregation in response to thrombin. The material secreted by rat platelets is heat labile and is sensitive to digestion with trypsin, suggesting that it is a protein. When assayed on 3T3 cells this material also stimulates DNA synthesis; however, the trypsin-sensitive activity is heat stable (100 degrees C, 10 min). These results indicate that rat platelets contain hepatotrophic activities which by virtue of their heat sensitivity are distinct from heat-stable platelet-derived growth factor (PDGF)-like mitogenic activities required by 3T3 cells for growth. It is possible that hepatotrophic platelet factors might be involved in mediating liver regeneration in the rat after partial hepatectomy.

[Biotransformation of monolinuron (N-(4-chlorophenyl)-N'-methyl-N'methoxyurea) in isolated perfused chicken liver]. / Biotransformation von Monolinuron (N-(4-Chlorphenyl)-N'-methyl-N'-methoxyharnstoff) in der isoliert perfundierten Hühnerleber.

The biotransformation of radioactively labelled monolinuron (N-(4-chloro[U-14C]phenyl)-N'-methyl-N'-methoxyurea) was studied in the isolated perfused liver of the chicken. After a 4-hr perfusion, 83.1% of the added radioactivity was recovered, 56.6% in the perfusion medium and 26.5% in the liver and bile. The fraction of radioactivity extractable from the perfusion medium into ethyl acetate amounted to 47.8% of the added dose. In addition to monolinuron, five breakdown products were identified in this extract, namely N-(4-chlorophenyl)-N'-hydroxymethyl-N'-methoxyurea, N-(4-chlorophenyl)-N'-methoxyurea, N-(4-chlorophenyl)-N'-methylurea, 4-chlorophenylurea and 4-chloroacetanilide. Of particular interest was the absence of arylhydroxylated monolinuron derivatives, since in monolinuron-metabolism studies in the laying hen 2-hydroxy-4-chlorophenylurea and 3-hydroxy-4-chlorophenylurea were both detected. This differing metabolism corresponds to earlier findings in the rat, in which arylhydroxylated breakdown products were detected only in in vivo studies and not in rat-liver perfusion. Possible reasons for the differing metabolism of monolinuron in vivo and in vitro are discussed.