John T. Johnson

A comparison of male rat and human urinary proteins: implications for human resistance to hyaline droplet nephropathy.

alpha 2u-Globulin (alpha G), the major urinary protein of sexually mature male rats, is a key determinant of susceptibility to hyaline droplet nephropathy (HDN) induced by a variety of hydrocarbons in male rats. Arguments against extrapolating renal toxicity and carcinogenicity data for HDN-inducing toxicants from male rats to risk assessment for humans rely on the observation that humans do not express alpha G. Yet, human serum and urine are known to contain proteins coded for by the same gene family that also controls alpha G synthesis in the rat. Therefore, to understand some of the quantitative and qualitative differences between proteins of human and male rat urine which confer apparent resistance to HDN in humans, urinary proteins of male F344 rats (ca. 3 months old) and normal human males were compared by cation exchange, gel filtration, SDS-PAGE, and partially identified by Western blotting. We observed that (1) the protein content of human urine is only 1% that of male rat urine; (2) human urinary proteins, recovered by (NH4)2SO4 precipitation followed by dialysis, are primarily of high (greater than or equal to 75 kDa) molecular weight (MW) with minor components of 12-66 kDa; (3) male rat urine has little high-MW protein, but is rich in alpha G (18.5 kDa); (4) at pH 5, the most cationic fraction of human urinary protein constituted only about 4% of the total while the analogous fraction of rat urine, containing alpha G, contained 26% of total urinary protein; and (5) cationic (at pH 5.0) human urinary proteins included small amounts of proteins, e.g., alpha 1-acid glycoprotein, and alpha 1-microglobulin, which are products of the gene family coding for alpha G in rat. Thus, although humans excrete trace amounts of proteins similar to alpha G, the very low protein content of human urine, the relatively small proportion of cationic to total proteins, and the high MW of the most abundant human urinary proteins form a biological basis for suggesting that humans are not at risk for the type of fuel and solvent hydrocarbon-induced nephropathy, and the sequelae of such nephropathy, observed in male rats.

Retention and clearance of inhaled submicron carbon black particles

Carbon black aerosols were used as a probe of the pulmonary retention and clearance of submicron particles. Male Fischer rats (COBS CD) were exposed for 20 h/d, 7 d/wk for 1, 3, or 6 wk to either 7 +/- 2 mg/m3 carbon black or filtered air. The submicron aerosol (mass median aerodynamic diameter, MMAD, 0.24 microns) was generated with a Wright dust feed-cyclone system. Lung and hilar lymph node particle burdens were determined immediately following the exposure and at preselected intervals up to 1 yr postexposure. After 1-, 3-, and 6-wk exposures, the lung burdens were 1.1 +/- 0.1, 3.5 +/- 0.2, and 5.9 +/- 0.1 mg, respectively. One year after a 1-, 3-, or 6-wk exposure, 8%, 46%, and 61% of the initial lung burden remained in the lungs. Initially, the hilar lymph nodes contained 0.2%, 0.9%, and 2.0% of the lung burdens in the 3 exposure groups, respectively. At 1 yr postexposure, particle translocation from the lungs led to a rise in lymph node burdens to 1%, 21%, and 27% of the initial lung burden. The retention of carbon black in both the lungs and lymph nodes combined was 9%, 67%, and 89% for the 1-, 3-, and 6-wk exposed animals. Lung clearance was modeled as a compartmental system consisting of four lung compartments and a regional lymph node compartment. The results from the model are similar for carbon black and diesel engine exhaust particles. However, the compartmental kinetics of carbon black differed in two ways: the deposition efficiency in the alveolar region was lower than that for diesel exhaust particles, and there was earlier transport of particles to the regional lymph nodes. These results showed that when lung burdens reached 0.8 mg, lung clearance was decreased by 50% and lymphatic transport of insoluble particles was increased.

Inhibitory effect of benzene metabolites on nuclear DNA synthesis in bone marrow cells

Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30-min incorporation of [3H]thymidine into DNA following a 30-min interaction with the cells in McCoys 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4-benzenetriol, hydroquinone, and p-benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 microM. In a cell-free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of [3H]thymidine triphosphate into DNA up to 24 microM but 1,2,4-benzenetriol and p-benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4-dithiothreitol (1 mM) in the cell-free assay system. Furthermore, when DNA polymerase alpha, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase I, which does not require an SH group for its catalytic activity, p-benzoquinone and 1,2,4-benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply that p-benzoquinone and 1,2,4-benzenetriol inhibited DNA polymerase alpha, consequently resulting in inhibition of DNA synthesis in both cellular and cell-free DNA synthetic systems. The present study identifies catechol, hydroquinone, p-benzoquinone, and 1,2,4-benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause.

Feasibility Study of Using Brine for Carbon Dioxide Capture and Storage from Fixed Sources

Abstract A laboratory-scale reactor was developed to evaluate the capture of carbon dioxide (CO2) from a gas into a liquid as an approach to control greenhouse gases emitted from fixed sources. CO2 at 5–50% concentrations was passed through a gas-exchange membrane and transferred into liquid media—tap water or simulated brine. When using water, capture efficiencies exceeded 50% and could be enhanced by adding base (e.g., sodium hydroxide) or the combination of base and carbonic anhydrase, a catalyst that speeds the conversion of CO2 to carbonic acid. The transferred CO2 formed ions, such as bicarbonate or carbonate, depending on the amount of base present. Adding precipitating cations, like Ca++, produced insoluble carbonate salts. Simulated brine proved nearly as efficient as water in absorbing CO2, with less than a 6% reduction in CO2 transferred. The CO2 either dissolved into the brine or formed a mixture of gas and ions. If the chemistry was favorable, carbonate precipitate spontaneously formed. Energy expenditure of pumping brine up and down from subterranean depths was modeled. We conclude that using brine in a gas-exchange membrane system for capturing CO2 from a gas stream to liquid is technically feasible and can be accomplished at a reasonable expenditure of energy.

Inhaled particle retention in rats receiving low exposures of diesel exhaust

To study the effects of a low concentration exposure on the retention and clearance of submicron particles from the lungs, we exposed male Fisher 344 rats to diesel exhaust diluted to 50 micrograms diesel exhaust particles (DP)/m3, 20 h/d, 7 d/wk for 52 wk. Lung burdens (amount of DP in lungs) and the alveolar macrophage burdens were measured up to 52 wk postexposure. By 1 yr postexposure at least 80% of the DP was eliminated from the lungs and similarly cleared from the lavaged pool of macrophages. The DP remaining in the lungs was observed in alveolar, parabronchial and paravascular maculae. In contrast to previous high concentration exposure studies, only trace amounts of particles were observed in the mediastinal lymph nodes. To study the concentration dependence of particle retention, rats were exposed to equivalent exposures of 18 d x mg DP/m3 delivered at 5700 micrograms/m3 for 3 d, 1600 micrograms/m3 for 12 d, 250 micrograms/m3 for 72 d, or 50 micrograms/m3 for 365 d. Higher lung and macrophage burdens were initially achieved with the brief, high concentration exposures. During the postexposure period, animals exposed to the higher concentrations cleared more of the lung burden. Exposure to lower concentrations resulted in higher long-term lung burdens. These results are consistent with a model of lung clearance in which the macrophage burden and the duration of exposure are both important to the formation of the maculae. In a brief high concentration exposure, the macrophage burden rises rapidly, but then declines rapidly. However, in longer low concentration exposures, the macrophage burden will not reach the same peak, but stays at intermediate levels during the exposure and stimulates a steady development of the lung maculae from particle-laden macrophages leaving the active pool of pulmonary phagocytes.

Modulation of Glucose Metabolism in Isolated Rat Hepatocytes by 1,1,1,2-Tetrafluoroethane

The thermodynamic behavior and lack of ozone-depleting potential of 1,1,1,2-tetrafluoroethane (R-134a) suggest it as a likely replacement for dichlorodifluoromethane (R-12), now used as the refrigerant in many air-conditioning systems. To further the presently incomplete toxicological analysis of R-134a, the effects of R-134a on cell viability and functional competence of glucose metabolism were evaluated in suspension cultures of hepatocytes derived from fed or fasted rats. R-134a concentrations up to and including 75% (750,000 ppm) in the gas phase of sealed culture flasks did not produce evidence of cytolethality (LDH leakage) following 2 hr of exposure; in contrast, halothane (1,1,1-trifluoro-2-bromo-2-chloroethane) caused cell death at a gas phase concentration of only 1250 ppm. In hepatocytes isolated from fed rats. R-134a at concentrations of 12.5 to 75% increased glycolysis (production of lactate + pyruvate) in a concentration-dependent manner; no effect was observed at 5%. At 25%, R-12 and 1,1,2,2-tetrafluoro-1,2-dichloroethane (R-114) were of equal potency to R-134a in stimulating glycolysis: 1,1,1,2,2-pentafluoro-2-chloroethane (R-115) depressed glycolysis slightly. Halothane, at concentrations as low as 300 ppm, markedly increased rates of glycolysis. Glucose production by hepatocytes of fed rats was decreased by R-134, R-12, and R-114 only at concentrations of 25% or more. On the other hand, halothane (greater than or equal to 300 ppm) potently decreased glucose production by hepatocytes. In cells isolated from livers of fasted rats, R-134a exposure inhibited gluconeogenesis in a concentration-dependent manner although this effect was not significant until R-134a concentrations reached 12.5%. Comparative potency studies showed that R-134a, R-12, or R-114 (25% gas phase) inhibited gluconeogenesis about equally while as little as 300 ppm halothane was effective and R-115 (25%) was without effect. Considering that the threshold for alteration of the rate of glucose metabolism in this in vitro paradigm is about 12.5% R-134a, we conclude that toxicologically significant alteration of glucose-linked bioenergetics is unlikely at the levels of R-134a exposure anticipated in workplace or environment.

Generation of diesel particles coated with polycyclic aromatic compounds—evidence suggesting that dinitropyrene formation is a collection artifact

Abstract A generator for complex aerosols which have diesel particulate cores and polycyclic aromatic compound (PAC) coatings, and a system for delivering these particles to laboratory animals by nose-only inhalation were developed. The core particles were from diesel exhaust. They were obtained after or without passing exhaust through a type of heated catalytic converter used in pre-1981 automobiles. A vapor pressure-temperature relationship, calculated from available thermodynamic data or derived experimentally, helped to control the amount of polycyclic aromatic compounds coated onto the freshly generated diesel particles. Results confirmed formation of dinitropyrene from the reaction of 1-nitropyrene coated particles with nitrogen dioxide during particle collection. This implies that the presence of highly mutagenic dinitropyrene in diesel particles is an artifact of collection.