Analysis of chloroethane toxicity and carcinogenicity including a comparison with bromoethane.

Chloroethane (CE) gas carcinogenicity is analyzed and determined from a National Toxicology Program (NTP) bioassay where an inhalation concentration of 15,000 ppm CE gas in air produced the highest incidence of an uncommon-to-rare tumor ever observed by the NTP. Persistently inhaled CE produces endometrial cancers in female mice. The first-tumor-corrected uterine endometrial incidence (I) in B6C3F1 mice is 90%, but no significant tumors occurred in F344 rats. The endometrial cancers dispersed by 1) migrating locally to the adjacent myometrium, 2) then migrating to the bloodstream by intravasation, 3) entering 17 distal organs by extravasation and adapting to the new tissue environment. Distal cancers retained sufficient endometrial cell features to be recognized at each metastatic site. CE produced one of the highest metastasis rates ever observed by NTP of 79%. Comparing CE with bromoethane (BE), a structural analogue, it was found that BE too produced rare murine endometrial cancers yielding the second highest NTP incidence rate of I = 58% with a similar high malignancy rate of 56%. Because of the historical rarity of endometrial tumors in the B6C3F1 mouse, both of these SAR haloethanes seem to be evoking a strong, related carcinogenic potential in B6C3F1 mice, but not in F344 rats. The question of whether humans are similar to mice or to rats is addressed here and in Gargas, et al., 2008. The powerful carcinogenesis caused by these halohydrocarbons may have been caused by excessive and metabolically unresolved acetaldehyde (AC) which is directly generated by Cyp2E1 in the oxidative elimination of CE. With >95% AC metabolic production, as predicted from pharmacokinetic (PK) studies depending on CE exposure, AC is the main elimination intermediate. AC is a known animal carcinogen and a strongly suspected human carcinogen. Also, CE causes incipient decreases of tissue essential glutathione pools [GSH] by Phase II conjugation metabolic elimination of CE (and BE), by glutantione transferase (GST), in most organs (except brain) exposed to high circulating CE and it metabolites. In three laboratories, an excessive stress reaction of hyperkinesis was observed only during 15,000 ppm gas exposure but not when the exposure ceased or when exposure was presented at 150 ppm. Test rodents other than the female mice did not exhibit a pattern of visible stress nor did they have a carcinogenic response to CE gas. Unremitting stress has been documented to contribute a feedback to the hypothalamus which stimulates the hypothalamic-pituitary-axis (HPA), which in turn, induces the adrenal glands. Because estrus and estrogen and progesterone levels were unaltered by CE gas, the adrenal over stimulation, causing high steroid output, may be the penultimate step in this extraordinary carcinogenic response. High adrenal production of corticosteroids could adversely promote endometrial cells to cancers in mice - a mechanism that has already been observed in humans.

Nitrobenzene carcinogenicity in animals and human hazard evaluation.

Nitrobenzene (NB) human cancer studies have not been reported, but animals studies have. Three rodent strains inhaling NB produce cancer at eight sites. B6C3F1 mice respond with mammary gland malignant tumors and male lung and thyroid benign tumors, F344/N male rats respond with liver malignant tumors and thyroid and kidney benign tumors, while females respond with endometrial polyps. Male Sprague-Dawley rats (CD strain) respond with liver benign tumors. NB is oxidized to various phenolic metabolites, while also being reduced in the cecum and systemically in the microsomes to nitrosobenzene (NOB), phenylhydroxylamine (PH), related free radicals, and aniline (AN). Based on structural and mechanistic similarities, NB compares with other animal and human carcinogenic nitroarenes and aromatic amines. Reduced NB first forms the nitroanion free radical, which can react with O2 to form superoxide O2*. Repeated NB dosing produces a persistent redox couple NOB<==>PH in red blood cells (RBCs) that generates met-Hb and expends NAD(P)H. NOB forms activated glutathione (GSH) conjugates. These biochemical effects may lead to critical redox imbalances and macromolecular binding. Known NB effects are hemosiderosis, methemoglobinemia, and anemia--and now dispersed cancer in rodents. On the basis of animal, metabolic and structure-activity studies, NB is determined to be a probable human carcinogen by any route of exposure.

Nitrobenzene potential human cancer risk based on animal studies.

Inhaled nitrobenzene (NB) in animals produces cancer at eight sites in three rodent strains. B6C3F1 mice respond with mammary gland malignant tumors and male lung and thyroid benign tumors, and F344/N male rats respond with liver malignant tumors and thyroid and kidney benign tumors, while females respond with endometrial polyps. Male Sprague-Dawley male rats (CD strain) respond with liver benign tumors. NB is oxidized to various phenolic metabolites, while also being reduced to nitrosobenzene (NOB), phenylhydroxylamine (PH), related free radicals, and aniline (AN) in the cecum by bacteria and in the body by the microsomes. In reduction, NB first forms the nitroanion free radical, which can react with O2 to form O2*-. Repeated NB dosing produces a persistent redox couple NOB<==>PH in red blood cells that generates met-Hb and expends NAD(P)H. NOB forms activated glutathione conjugates. These biochemical effects may lead to critical redox imbalances and macromolecular binding. Known effects are hemosiderosis, methemoglobinemia, and anemia--and now dispersed cancer in rodents. Based on structural and mechanistic similarities, NB compares with other animal and human carcinogenic nitroarenes and aromatic amines. The cancer hazard evaluation of NB is that it is a probable human carcinogen by any route of exposure. The maximum response is in F344/N male rats which is used for dose-response modelling. The model to estimate the upper 95% confidence limit (UCL95%) of NB human carcinogenicity is a no-threshold, linear low-dose, and multistaged animal model (LMS). The UCL95% of cancer slope is estimated to be 0.11(6) mg/kg/day (mkd). At de minimus risk (1:10(6)), the virtually safe dose (VSD) is estimated to be 9.1 ng/kg/day (nkd).

Gap junction function and cancer.

Gap junctions (GJs) provide cell-to-cell communication of essential metabolites and ions. GJs allow tissues to average responses, clear waste products, and minimize the effects of xenobiotics by dilution and allowing steady-state catabolism. Many chemicals can adversely affect the membrane GJ assembly causing reversible alterations in GJ intercellular communication. During toxicity essential metabolites, ions, and regulators are not shared homeostatically throughout a tissue community. Alterations in metabolic circuits are thought to interrupt organ integration. Persistent GJ perturbation can cause chronic effects (e.g., cancer), and many tumor promoters inhibit GJ intercellular communication. Liver precancerous foci intracommunicate (but at a reduced level) and intercommunicate improperly (or not at all) across the foci boundary to normal cells. In time, foci can become less regulated and more isolated within the tissue. GJs remain reduced quantitatively in the tumor progression stage and may be qualitatively altered in metastasis since connections are made between the primary tumor cells and foreign host cells at the secondary metastatic site. Cell sorting and binding mechanisms by the cell adhesion molecules and integrins may also be altered at secondary sites. This may allow the relocation of primary tumor cells and nurturance via GJs at the secondary site.

Ethylene dibromide residues in biscuits and commercial flour.

Flour and biscuit samples from a school lunch program were analyzed for ethylene dibromide (EDB). Flour samples were extracted with hexane at room temperature with maximum extraction of EDB in 4 days. Biscuits were extracted by steam distillation with hexane; optimum recoveries were obtained by a triple extraction of the sample. Recoveries of EDB from flour and biscuits ranged from 85 to 103% as determined by gas-liquid chromatography on a 15% OV-17 column and a 63Ni electron capture detector. Random samples were confirmed by gas chromatography-mass spectrometry. From less than 8 ppb to 4 ppm EDB were determined in flour and less than 0.5 ppb to 260 ppb in biscuits. Possible sources for the higher values are discussed.

Secondary structure of ovalbumin messenger RNA.

The secondary structure of highly purified ovalbumin mRNA was studied by automated thermal denaturation techniques and the data were subjected to computer processing. Comparative studies with 20 natural and synthetic model nucleic acids suggested that the secondary structure of ovalbumin mRNA possesses the following features: the extent of base pairing of ovalbumin mRNA is similar to that found in tRNAs or ribosomal RNAs; the secondary structure of ovalbumin mRNA is more thermolabile than any of the model compounds tested, including the copolymer poly(A-U); ovalbumin mRNA does not have extensive G-C rich stems as found in tRNAs or ribosomal RNAs; the base composition of the double-stranded regions reveals 54% G-C residues which was significantly higher than that noted in the whole molecule (approximately 41.5% G-C). The presence of 46% A-U pairs in short stems of about five base pairs would have a very large destabilizing effect on the secondary structure of ovalbumin mRNA. However, at 0.175 M monovalent cations and 36 degrees C most of the secondary structure of ovalbumin mRNA is preserved. These data suggest that the double-stranded regions in ovalbumin mRNA are of sufficient length to provide the necessary stability for maintaining the open loop regions in an appropriate conformation which may be required for the biological function of ovalbumin mRNA. Furthermore, the lability of the double-stranded regions in ovalbumin mRNA may also be important for the biological function of this mRNA.

Determination of secondary structure in rabbit globin messenger RNA by thermal denaturation.

The secondary structure of highly purified globin messenger RNA has been investigated by alkaline hydrolysis, nuclease digestion, and thermal denaturation. The thermal denaturation properties of globin messenger have been compared to poly(U), poly (A), and a synthetic random sequence RNA copolymer. From these studies it is concluded that globin mRNA contains considerable secondary structure and that the amount of helical structure is greater than that which occurs with a random sequence polyribonucleotide. Globin mRNA contains, by comparison to the secondary structures of native DNA, tRNAs, or 18S rRNA, helices with involve 55-62% of the bases or 58-68% if a correction is made for the 3'-terminal poly(A) segment. The helices of globin mRNA appear to be unique as differences in the NaCl stabilization of this RNA have been noted when compared to other naturally ooccurring and synthetic RNAs. Comparison of the hyperchromicity maxima, obtained at 260 and 280 nm for globin mRNA and 18S rRNA, indicates that the helices of the two RNAs contain similar numbers of G-C base pairs. Differential analysis of NaCl stabilization curves indicate three discrete thermally denaturable helix types in globin mRNA.

Preparation and preliminary characterization of purified ovalbumin messenger RNA from the hen oviduct.

Preparation of milligram amounts of purified ovalbumin mRNA was accomplished by a sequential combination of precise sizing techniques with the selective purification of the poly(A) containing RNA by either affinity chromatography or adsorption to nitrocellulose filters. Several new techniques were applied to the purification of ovalbumin mRNA including Sepharose 4B chromatography and agarose gel electrophoresis in the presence of 6 M urea at pH 3.5. All the procedures used were adapted on a preparative sacle to the fractionation of large quantities of RNA. The purity of the ovalbumin mRNA was assessed by several independent criteria. (1) Purified ovalbumin mRNA migrated as a single band during both agarose-urea and formamide-polyacrylamide gel electrophoresis at pH 3.5 and 7.4, respectively. A single absorbance peak containing all of the ovalbumin mRNA activity was also found using linear formamide-sucrose gradients. (2) Determination of both total mRNA activity and ovalbumin mRNA activity in the wheat germ cell-free translation assay revealed that 92% of the total peptides synthesized were specifically immunoprecipitable with an ovalbumin antiserum. (3) Analysis of the total peptides synthesizied in the wheat germ assay by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of a single radioactive peak that corresponded exactly to a specifically immunoprecipitable ovalbumin standard. Thus, based on these observations ovalbumin mRNA appears to be greater than 95% pure. A preliminary estimation of the molecular weight of purified ovalbumin mRNA by formamide-containing sucrose gradients yielded a value of 520,000 or approximately 1600 nucleotides. This value was considerably less than the value of 900,000 obtained by gel electrophoresis under denaturing conditions. Analysis of the poly(A) content by a hybridization assay with (3H)poly(U) revealed the presence of a poly(A) region containing approximately 70 adenosine residues. Thus, the size of the ovalbumin mRNA is considerably greater than that required to code for a protein of 387 amino acids. The availability of large quantities of purified ovalbumin mRNA should now permit a more thorough analysis of its physical and chemical properties.